Rsv virus-like particles and methods of use thereof

ABSTRACT

The present disclosure relates to virus-like particles and vaccine compositions for inducing immunity and preventing respiratory syncytial virus (RSV) infection. Specifically, the disclosure provides virus like-particles (VLPs) for use in inducing immunity to respiratory syncyhial virus (RSV) infections or symptoms thereof, wherein the VLP comprising a respiratory RSV matrix protein (M) and an RSV M2-1 protein, a glycoprotein (G), a fusion protein (F), and/or a phosphoprotein (P).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/751,975 filed Oct. 29, 2018, the disclosure ofwhich is expressly incorporated herein by reference.

FIELD

The present disclosure relates to virus-like particles and uses thereof.

BACKGROUND

Respiratory Syncytial Virus (RSV) was quickly recognized as an importantpediatric pathogen after its discovery in the 1950s. It causes upper andlower respiratory tract infections including bronchitis, bronchiolitis,and pneumonia. Most children are infected by 2 years of age. However,since its infection provides incomplete protection, RSV infectsthroughout life with the elderly and persons with chronic cardiac orpulmonary disease, or immune compromising conditions at higher risk forsevere complications. It is estimated that globally there are more than33 million episodes of RSV infections and 95,000-150,000 RSV deaths,mostly in developing countries, in children <5 years of age. RSV-relateddeaths are rare in the United States; it is, however, responsible for anestimated 60,000-170,000 hospitalizations each year in children <5 yearsof age. Also, infants hospitalized with RSV infection are prone to laterdevelopment of obstructive airway diseases and asthma. Its substantialglobal disease burden has made RSV a high priority for vaccine andanti-viral drug development. There are, however, no effective anti-viraldrugs or vaccines yet available. Therefore, what is needed is a vaccinefor inducing protective immunity to RSV infection. The compositions andmethods disclosed herein address these and other needs.

SUMMARY

Disclosed herein are virus like-particles (VLPs) for use in inducingimmunity to respiratory syncytial virus (RSV) infections or symptomsthereof.

In some aspects, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein and an RSV M2-1protein.

In some embodiments, the VLP comprises one or more additional RSVproteins. In some embodiments, the VLP comprises an RSV F protein. Insome embodiments, the RSV F protein is selected from a group consistingof a pre-fusion form of the RSV F protein, a post-fusion form of the RSVF protein, and a carbonyl terminal portion of the RSV F protein. In someembodiments, the RSV F protein comprises a sequence selected from thegroup consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 37. In someembodiments, the carbonyl terminal portion of the RSV F proteincomprises a sequence of SEQ ID NO: 32.

In some embodiments, the VLP comprises an RSV G protein. In someembodiments, the RSV G protein is from RSV group A or RSV group B. Insome embodiments, the RSV G protein comprises a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

In some embodiments, the VLP comprises a recombinant RSV G protein. Insome embodiments, the recombinant RSV G protein comprises atransmembrane domain of an RSV G protein and a central conserved domainof an RSV G protein.

In some aspects, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein, an RSV Pprotein, an RSV F protein, and an RSV G protein.

In some embodiments, the RSV F protein is selected from a groupconsisting of a pre-fusion form of the RSV F protein, a post-fusion formof the RSV F protein, and a carbonyl terminal portion of the RSV Fprotein. In some embodiments, the RSV F protein comprises a sequenceselected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQID NO: 29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:37. In some embodiments, the carbonyl terminal portion of the RSV Fprotein comprises a sequence of SEQ ID NO: 32.

In some embodiments, the RSV G protein is from RSV group A or RSV groupB. In some embodiments, the RSV G protein is from RSV group A and RSVgroup B. In some embodiments, the RSV G protein comprises a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

In some embodiments, the RSV G protein is a recombinant RSV G protein.In some embodiments, the VLP comprises a recombinant RSV G protein. Insome embodiments, the recombinant RSV G protein comprises atransmembrane domain of the RSV G protein and a central conserved domainof the RSV G protein.

In some aspects, disclosed herein is a vaccine comprising the VLP of anypreceding aspect. In some embodiments, the vaccine further comprises anadjuvant.

In some aspects, disclosed herein is a method of inducing animmunological response to RSV infection or at least one symptom thereofin a subject, comprising administering one or more effective doses ofthe vaccine of any preceding aspect. In some embodiments, the one ormore effective doses of the vaccine are administered to the subject viaa route that is selected from the group consisting of an intramuscularroute, a subcutaneous route, an intradermal route, an oraladministration, a nasal administration, and inhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIGS. 1A-1C show generation and expression of F and G on RSV VLPs. 293Fcell line expressing RSV gene M, F (or F_(t)), G (or G_(P)) and P orM2-1 were induced for 72 h in 2 μg/ml doxycycline. Cells were harvestedand low centrifugation performed to separate cells and VLPs-containingsupernatant. VLPs were filtered through 0.45 μm filter to clear celldebris, layered on top of 20% sucrose and subjected to centrifugation at12,200×g for 2 h, 4° C. VLP pellets were resuspended in sterile PBS andsubjected to centrifugation through a 20-60% sucrose gradient at11,000×g for 12 h, 4° C. A total of 10 fractions were collected andanalyzed by immunoblotting using 3D3 (human anti-G antibody),motavizumab (human anti-F antibody), and rabbit serum anti-M antibody.FIG. 1A shows VLPs MFGP and MFGM2-1. FIG. 1B shows VLPs MFG_(P)P andMFG_(P)M2-1. FIG. 1C shows MGP, MFP, and MF_(t)GP VLPs. G_(P), truncatedG: aa 1-86+155-206. F_(t), truncated F: aa 496-574.

FIGS. 2A and 2B show that glycoproteins are visualized as spikes on VLPsby negative stain electron microscopy. FIG. 2A shows that a 3 μl aliquotcontaining the diluted sample was applied for 1 min onto aformvar/carbon coated, 300 mesh-copper grid that has been glowdischarged for 30 sec, then negatively stained with 0.75% freshly-madeuranyl formate on ice for 1 min. Data were collected using a FEI T20electron microscope operating at 200 kV (pixel size 1.101 A, totalelectron dose is 54 electrons/A square)”. FIG. 2B shows that MFGP VLPswere labeled with 3D3 or motavizumab followed by incubating withgold-labeled anti human secondary antibody.

FIGS. 3A-3C show that immunized animals generate serum anti-G and anti-Fantibodies. FIG. 3A shows schematic schedule of animal experiments. FIG.3B shows that sera from immunized animals (diluted 1:200) were used inbinding ELISA to immobilize 293F cell lysate containing Ga, Gb, or Fantigen. After blocking, plates were incubated with goat anti mouseIgG-HRP secondary antibody. OPD substrate was used to develop reactionand absorbance at 490 nm was read. FIG. 3C shows that sera fromimmunized animals were heat inactivated at 56° C. for 30 min followed by2-fold serial dilution in triplicates. The dilutions were incubated with100 TCID50 of RSV A2 virus for 1 h at RT. The mixtures were thentransferred to monolayer HEp-2 cell and incubated for 1 h at 37° C. in5% CO₂. 5% FBS+MEM media was added to the cells followed by incubationfor 72 h at 37° C. in 5% CO₂. Cells were fixed and ELISA was performedusing goat anti-RSV antibody and HPR-conjugated donkey anti-goatsecondary antibody. Reaction was developed by OPD and absorbance read at490 nm. Neutralizing titers were calculated using Reed-Muench method.

FIG. 4 shows that immunized animals have significant less lung viraltiter. Lungs from immunized animals were homogenized as described inmaterials and methods. Aliquots stored at −80° C. were thawed and totalRNA was extracted from lung. RNAs were then reverse transcribed intocDNAs. These were used as templates in RT-PCR using CYBR green and apair of RSV matrix protein M specific primers as described. In parallel,similar reactions were performed using a pair of β-actin specificprimers as controls. Results were expressed as relative amount of RSV Mcompared to β-actin. * p<0.05.

FIGS. 5A and 5B shows that immunized animals had significant less lungmucin. Female BALB/c mice (4-6 weeks) were divided in 7 groups (n=4),immunized, and challenged as summarized in table 2. Lungs werecollected, fixed, and stained with Periodic-acid Schiff (PAS) stainingas described in materials and methods. The slides were analyzed byAperio ImageScope software and scored blindly on a 0-4 scale andsubsequently converted to a 0-100% histopathology scale. FIG. 5A showsrepresentative images from corresponding groups. FIG. 5B showsquantitative data converted from histopathology scale. * p<0.05, **p<0.01.

FIG. 6 shows domains of Ga protein, Gb protein, Fpost protein and Fpreprotein and combination of F and G expression of RSV VLPs usingplatforms M+P and M+M2-1.

FIG. 7 shows G and/or F expression of RSV VLPs. 293F cells expressingVLPs were induced for 72 h. Cell supernatants were collected,centrifuged, purified through 0.45p m filter and 20% sucrose at 10,000×gfor 2 h, 4° C. Pellets resuspended in PBS as well as solubilized cellswere analyzed by immunoblotting with anti-G (3D3) and anti-F(motavizumab) antibodies. S: supernatant; C, cells.

FIG. 8 shows that glycoproteins are visualized as spikes on VLPs bynegative stain electron microscopy. A 3 μl aliquot containing thediluted sample was applied for 1 minute onto a formvar/carbon coated,300 mesh-copper grid that has been glow discharged for 3 seconds, thennegatively stained with 0.75% freshly-made uranyl formate on ice for 1minute. Data were collected using a FEI T20 electron microscopeoperating at 200 kV (pixel size 1.101 A, total electron dose is 54electrons/A square).

FIG. 9 shows a schematic of examples of RSV F proteins. DS-Cav1: thisstructure-based design generates a stabilized prefusion F structure thatretains antigenic site Ø (recognized by the prefusion specific antibodyD25). This is achieved by creating a double-mutation at amino acidsS155C and S290C forming a stable F trimer. That combines with S190F andV207L hydrophobic pair mutations that fill a cavity in the structurecreating DS-Cav1. SC-DM and SC-TM are both designed to have a shortlinker between F1 and F2 subunits of the F protein. Further mutations inthe F secondary structure to limit transformation from prefusion topostfusion conformation at N67I and S215P creates SC-DM. Additionalmutation at E487Q to minimize negative repulsion generates SC-TM.

DETAILED DESCRIPTION

RSV is a single-stranded, negative sense RNA virus belonging toParamyxoviridae family and Pneumoviridae subfamily with two distinctantigenic groups, A and B. The RSV genome of approximately 15.2 kbincludes ten genes that encode for eleven proteins. Three proteins, thefusion (F), attachment (G), and small hydrophobic (SH) are expressed onthe virion envelop. The F and G proteins are the only proteins shown toinduce effective neutralizing antibodies and longer-term protectiveimmunity. The F protein is more conserved among RSV strains and inducescross-protective immunity and is most effective at inducing neutralizingantibodies. The SH protein does induce some protection likely through Fcreceptor-mediated activity such as antibody dependent cellularcytotoxicity or complement activation. Most neutralizing antibodies inhuman serum specimens are against the pre-fusion form of F andpre-fusion F is currently a prime candidate for RSV vaccines. The Gprotein, though eliciting less potent neutralizing antibody, has beenshown to be an important factor for RSV disease pathogenesis making italso a candidate for inclusion in an RSV vaccine. The G proteinstructure consists of a conserved region that contains a CX3C chemokinemotif that enables binding to the CX3C chemokine receptor, CX3CR1, andhas some activities similar to the CX3C chemokine fractalkine. The Gprotein induces disease causing inflammatory responses that can beinhibited by blocking G binding to CX3CR1 with passive administration ofan anti-G monoclonal antibody, G peptide vaccine induced antibodies, orby mutating the CX3C motif. Thus, a vaccine that induces both anti-F andanti-G antibodies can decrease disease by both decreasing viralreplication and producing an anti-inflammatory effect.

Despite over 60 years of research, no effective vaccine or antiviraldrug is available. Studies, however, show most neutralizing antibodiesproduced after RSV infection are against the surface fusion F andattachment G glycoproteins, thus making these the prime candidates forvaccines. In fact, antibodies induced by the F protein reduce viraltiters. Antibodies induced by the G protein are less effective atreducing viral titers than those induced by F but reduce inflammationand disease more effectively. Among different strategies for RSVvaccines, virus-like particles (VLPs) are safe, immunogenic, and used inlicensed human vaccines.

Disclosed herein is a vaccine that includes both the F and G proteinsutilizing M based virus-like particles (VLPs). An RSV-based vaccineplatform is optimal since all components can contribute tovaccine-induced immunity and the protein structures in the VLP betterreflect natural structures. A VLP that better reflects the structure inthe virus can induce a more effective immune response and provide abetter model for structural studies. RSV VLPs have previously beendeveloped using the Newcastle disease, influenza, or bacterial phage P22platforms with the RSV F and/or G proteins or M and M2 proteins.

Disclosed herein are novel RSV VLPs with F and/or G proteins using anRSV platform with M plus P or M plus M2-1. Since M2-1 is important forstructural stability of RSV, M2-1 can also contribute to VLP formation.The data herein show that an RSV-based vaccine platform can be used toefficiently form RSV VLPs with either M plus P or M plus M2-1 with Fand/or G proteins. Unexpectedly, the inventors found that the G proteinwas only efficiently incorporated into these VLPs in the presence of theintracellular and transmembrane domains of F.

Described herein are virus like-particles (VLPs) and methods forinducing immunity to respiratory syncytial virus (RSV) infections orsymptoms thereof. In one aspect, disclosed herein is a virus likeparticle (VLP) comprising a respiratory syncytial virus (RSV) M proteinand an RSV M2-1 protein, wherein the VLP further comprises an RSV Fprotein and/or an RSV G protein. In one aspect, disclosed herein is avirus like particle (VLP) comprising a respiratory syncytial virus (RSV)M protein, an RSV P protein, an RSV F protein, and an RSV G protein.

Reference will now be made in detail to the embodiments of theinvention, examples of which are illustrated in the drawings and theexamples. This invention may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs.

Terminology

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicant desires that the following terms be given the particulardefinition as defined below.

As used herein, the article “a,” “an,” and “the” means “at least one,”unless the context in which the article is used clearly indicatesotherwise.

The term “comprising” and variations thereof as used herein is usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms. Although the terms “comprising” and“including” have been used herein to describe various embodiments, theterms “consisting essentially of” and “consisting of” can be used inplace of “comprising” and “including” to provide for more specificembodiments and are also disclosed.

As used herein, the terms “may,” “optionally,” and “may optionally” areused interchangeably and are meant to include cases in which thecondition occurs as well as cases in which the condition does not occur.Thus, for example, the statement that a formulation “may include anexcipient” is meant to include cases in which the formulation includesan excipient as well as cases in which the formulation does not includean excipient.

The terms “about” and “approximately” are defined as being “close to” asunderstood by one of ordinary skill in the art. In one non-limitingembodiment, the terms are defined to be within 10%. In anothernon-limiting embodiment, the terms are defined to be within 5%. In stillanother non-limiting embodiment, the terms are defined to be within 1%.

As used herein the term “adjuvant” refers to a compound that, when usedin combination with a specific immunogen in a formulation, will augmentor otherwise alter or modify the resultant immune response. Modificationof the immune response includes intensification or broadening thespecificity of either or both antibody and cellular immune responses.Modification of the immune response can also mean decreasing orsuppressing certain antigen-specific immune responses.

A “composition” is intended to include a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

As used herein, the term “virus-like particle” (VLP) refers to astructure that in at least one attribute resembles a virus but which hasnot been demonstrated to be infectious. In general, virus-like particleslack a viral genome and, therefore, are noninfectious. In addition,virus-like particles can often be produced in large quantities byheterologous expression and can be easily purified.

As used herein, the term “vaccine” refers to a formulation whichcontains VLPs of the present invention, which is in a form that iscapable of being administered to a subject and which induces aprotective immune response sufficient to induce immunity to preventand/or ameliorate an infection and/or to reduce at least one symptom ofan infection and/or to enhance the efficacy of another dose of VLPs.Typically, the vaccine comprises a conventional saline or bufferedaqueous solution medium in which the composition of the presentinvention is suspended or dissolved. In this form, the composition ofthe present invention can be used conveniently to prevent, ameliorate,or otherwise treat an infection. Upon introduction into a host, thevaccine is able to provoke an immune response including, but not limitedto, the production of antibodies and/or cytokines and/or the activationof CD8+ T cells, antigen presenting cells, CD4+ T cells, dendritic cellsand/or other cellular responses.

As used herein an “effective dose” generally refers to that amount ofVLPs or vaccines of the invention sufficient to induce immunity, toprevent and/or ameliorate an infection or to reduce at least one symptomof an infection and/or to enhance the efficacy of another dose of a VLPor a vaccine. An effective dose may refer to the amount of VLPs orvaccines sufficient to delay or minimize the onset of an infection or asymptom of an infection. An effective dose may also refer to the amountof VLPs or vaccines that provides a therapeutic benefit in the treatmentor management of an infection a symptom of an infection. Further, aneffective dose is the amount with respect to VLPs or vaccines of theinvention alone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of an infection. Aneffective dose may also be the amount sufficient to enhance a subject's(e.g., a human's) own immune response against a subsequent exposure toan infectious agent. Levels of immunity can be monitored, e.g., bymeasuring amounts of neutralizing secretory and/or serum antibodies,(e.g., by plaque neutralization, complement fixation, enzyme-linkedimmunosorbent, or microneutralization assay) and/or responses ofvirus-specific CD4+ T cells and CD8+ T cells, and/or responses of otherimmune cells. In the case of a vaccine, an “effective dose” is one thatprevents or reduces disease and/or prevents or reduces the severity ofsymptoms.

As used herein, the term “effective amount” refers to an amount of VLPsor vaccines comprising VLPs necessary or sufficient to realize a desiredbiologic effect. An effective amount of the composition would be theamount that achieves a selected result, and such an amount could bedetermined as a matter of routine experimentation by a person skilled inthe art. For example, an effective amount for preventing, treatingand/or ameliorating an infection could be that amount necessary to causeactivation of the immune system, resulting in the development of anantigen specific immune response upon exposure to VLPs or vaccinescomprising VLPs of the invention. The term is also synonymous with“sufficient amount.”

An “immunological response” or “immunity” to a composition or vaccine isthe development in the host of a cellular and/or antibody-mediatedimmune response to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredviral titer in the infected host.

As used herein the term “protective immune response”, “protectiveresponse”, or “protective immunity” refers to an immune responsemediated by antibodies against an infectious agent, which is exhibitedby a vertebrate (e.g., a human), that prevents or ameliorates aninfection or reduces at least one symptom thereof. VLPs of the inventioncan stimulate the production of antibodies that, for example, neutralizeinfectious agents, block infectious agents from entering cells, blockreplication of said infectious agents, and/or protect host cells frominfection and destruction. The term can also refer to an immune responsethat is mediated by T cells, B cells, and/or other white blood cellsagainst an infectious agent, exhibited by a vertebrate (e.g., a human),that prevents or ameliorates RSV infection or reduces at least onesymptom thereof.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In someembodiments, the subject is a human.

“Pharmaceutically acceptable carrier” (sometimes referred to as a“carrier”) means a carrier or excipient that is useful in preparing apharmaceutical or therapeutic composition that is generally safe andnon-toxic, and includes a carrier that is acceptable for veterinaryand/or human pharmaceutical or therapeutic use. The terms “carrier” or“pharmaceutically acceptable carrier” can include, but are not limitedto, phosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.

As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia, Pa., 2005.Examples of physiologically acceptable carriers include saline,glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, andbuffers with other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™ (ICI, Inc.; Bridgewater, N.J.), polyethyleneglycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.). To provide forthe administration of such dosages for the desired therapeutictreatment, compositions disclosed herein can advantageously comprisebetween about 0.1% and 99% by weight of the total of one or more of thesubject compounds based on the weight of the total composition includingcarrier or diluent.

As used herein, the terms “treating” or “treatment” of a subjectincludes the administration of a drug to a subject with the purpose ofcuring, healing, alleviating, relieving, altering, remedying,ameliorating, improving, stabilizing or affecting a disease or disorder,or a symptom of a disease or disorder. The terms “treating” and“treatment” can also refer to reduction in severity and/or frequency ofsymptoms, elimination of symptoms and/or underlying cause, andimprovement or remediation of damage.

“Therapeutically effective amount” or “therapeutically effective dose”of a composition (e.g. a VLP or a vaccine comprising a VLP) refers to anamount that is effective to achieve a desired therapeutic result. Insome embodiments, a desired therapeutic result is the prevention of anRSV infection and/or a symptom thereof. In some embodiments, a desiredtherapeutic result is the treatment of an RSV infection and/or a symptomthereof. Therapeutically effective amounts of a given therapeutic agentwill typically vary with respect to factors such as the type andseverity of the disorder or disease being treated and the age, gender,and weight of the subject. The term can also refer to an amount of atherapeutic agent, or a rate of delivery of a therapeutic agent (e.g.,amount over time), effective to facilitate a desired therapeutic effect,such as coughing relief. The precise desired therapeutic effect willvary according to the condition to be treated, the tolerance of thesubject, the agent and/or agent formulation to be administered (e.g.,the potency of the therapeutic agent, the concentration of agent in theformulation, and the like), and a variety of other factors that areappreciated by those of ordinary skill in the art. In some instances, adesired biological or medical response is achieved followingadministration of multiple dosages of the composition to the subjectover a period of days, weeks, or years.

The term “nucleic acid” as used herein means a polymer composed ofnucleotides, e.g. deoxyribonucleotides or ribonucleotides.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “oligonucleotide” denotes single- or double-stranded nucleotidemultimers of from about 2 to up to about 100 nucleotides in length.Suitable oligonucleotides may be prepared by the phosphoramidite methoddescribed by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862(1981), or by the triester method according to Matteucci, et al., J. Am.Chem. Soc., 103:3185 (1981), both incorporated herein by reference, orby other chemical methods using either a commercial automatedoligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotidesare referred to as “double-stranded,” it is understood by those of skillin the art that a pair of oligonucleotides exist in a hydrogen-bonded,helical array typically associated with, for example, DNA. In additionto the 100% complementary form of double-stranded oligonucleotides, theterm “double-stranded,” as used herein is also meant to refer to thoseforms which include such structural features as bulges and loops,described more fully in such biochemistry texts as Stryer, Biochemistry,Third Ed., (1988), incorporated herein by reference for all purposes.

The term “polynucleotide” refers to a single or double stranded polymercomposed of nucleotide monomers.

The term “polypeptide” refers to a compound made up of a single chain ofD- or L-amino acids or a mixture of D- and L-amino acids joined bypeptide bonds.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher identity over a specified region whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about10 amino acids or 20 nucleotides in length, or more preferably over aregion that is 10-50 amino acids or 20-50 nucleotides in length. As usedherein, percent (%) nucleotide sequence identity is defined as thepercentage of amino acids in a candidate sequence that are identical tothe nucleotides in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410).These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01.

The term “engineered” or “recombinant” means a polynucleotide orpolypeptide of semisynthetic, or synthetic origin that either does notoccur in nature or is operably linked to another polynucleotide in anarrangement not found in nature.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, operably linked nucleic acids (e.g. enhancers and codingsequences) do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. In some embodiments, a promoter is operablylinked with a coding sequence when it is capable of affecting (e.g.modulating relative to the absence of the promoter) the expression of aprotein from that coding sequence (i.e., the coding sequence is underthe transcriptional control of the promoter).

A “vector” refers to a recombinant DNA plasmid, bacteriophage, or virusthat comprises a heterologous polynucleotide to be delivered to a targetcell, either in vitro or in vivo. The heterologous polynucleotide maycomprise a sequence of interest for purposes of prevention or therapy,and may optionally be in the form of an expression cassette. As usedherein, a vector may be able to but does not need to be capable ofreplication in the ultimate target cell or subject. The term includesvectors for cloning as well as viral vectors.

The term “gene” or “gene sequence” refers to the coding sequence orcontrol sequence, or fragments thereof. A gene may include anycombination of coding sequence and control sequence, or fragmentsthereof. Thus, a “gene” as referred to herein may be all or part of anative gene. A polynucleotide sequence as referred to herein may be usedinterchangeably with the term “gene”, or may include any codingsequence, non-coding sequence or control sequence, fragments thereof,and combinations thereof. The term “gene” or “gene sequence” includes,for example, control sequences upstream of the coding sequence (forexample, the ribosome binding site).

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

Virus-Like Particles and Vaccines

Respiratory Syncytial Virus (RSV) is the leading cause of severebronchiolitis in infants and young children and a high priority forvaccine development. Disclosed herein are virus-like particles (VLPs)and the use thereof for inducing immune responses to RSV infection or atleast one symptom thereof in a subject. VLPs are used herein forvaccines since they are immunogenic and are safe to human. The presentdisclosure provides RSV VLPs with F and/or G using an RSV platform withM plus P or M plus M2-1, and uses thereof for inducing protective immuneresponses.

The respiratory syncytial virus (RSV), a member of the speciesorthopneumovirus of Orthopneumovirus genus, is a syncytial virus thatcauses respiratory tract infections. RSV has a single stranded negativesense RNA genome which is approximately 15.2 Kb long. RSV has beenclassified into two groups (group A and group B, or termed as “strain Aand strain B” herein) on the basis of genetic and antigenicheterogeneity. The two major glycoprotein on the surface of the RSVvirion are the attachment glycoprotein (G) and fusion protein (F). G isinvolved in attachment of virion to the host cells, and F cause thevirion membrane to fuse with cell membrane. In addition, four of theviral genes code for intracellular proteins that are involved in genometranscription, replication, and particle budding, namely N(nucleoprotein), P (phosphoprotein), M (matrix protein), and L (“large”protein, containing the RNA polymerase catalytic motifs).

In some aspects, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein and an RSV M2-1protein.

As used herein, the term “RSV Matrix” or “RSV M” protein refers to anRSV protein that, when expressed in a host cell, induces formation ofVLPs. An example of an RSV M protein is represented by SEQ ID NO: 39 orSEQ ID NO: 41. The term also comprises any variants, derivatives and/orfragments of RSV M protein that, when expressed in a host cell, inducesformation of VLPs. In some embodiments, the M polypeptide comprises thesequence set forth in SEQ ID NO: 39 or SEQ ID NO: 41, or sequence havingat or greater than about 80%, about 85%, about 90%, about 95%, about98%, or about 99% homology with SEQ ID NO: 39 or SEQ ID NO: 41, or apolypeptide comprising a portion of SEQ ID NO: 39 or SEQ ID NO: 41. Insome embodiments, the M polypeptide comprises the sequence set forth inSEQ ID NO: 39. In some embodiments, the M polypeptide comprises thesequence set forth in SEQ ID NO: 41. The term also encompassesnucleotide sequences which encode for RSV M and/or any variants,derivatives and/or fragments thereof that when transfected (or infected)into a host cell will express RSV M protein and induce formation ofVLPs. In some embodiments, the nucleotide sequence encoding Mpolypeptide comprises the sequence set forth in SEQ ID NO: 40 or SEQ IDNO: 42, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with SEQ ID NO:40 or SEQ ID NO: 42, or a polynucleotide comprising a portion of SEQ IDNO: 40 or SEQ ID NO: 42.

As used herein, the term “RSV M2-1 protein” or “M2-1 protein” refers toa cofactor of the RSV viral RNA polymerase complex and functions as atranscriptional processivity and antitermination factor. An example ofan RSV M2-1 protein is represented by SEQ ID NO: 43 or SEQ ID NO: 45.The term also comprises any variants, derivatives and/or fragments ofRSV M2-1 protein that, when expressed in a host cell, induces formationof VLPs. In some embodiments, the M2-1 polypeptide comprises thesequence set forth in SEQ ID NO: 43 or SEQ ID NO: 45, or sequence havingat or greater than about 80%, about 85%, about 90%, about 95%, about98%, or about 99% homology with SEQ ID NO: 43 or SEQ ID NO: 45, or apolypeptide comprising a portion of SEQ ID NO: 43 or SEQ ID NO: 45. Insome embodiments, the M2-1 polypeptide comprises the sequence set forthin SEQ ID NO: 43. In some embodiments, the M2-1 polypeptide comprisesthe sequence set forth in SEQ ID NO: 45. The term also encompassesnucleotide sequences which encode for RSV M2-1 and/or any variants,derivatives and/or fragments thereof that when transfected (or infected)into a host cell will express RSV M2-1 protein and induce formation ofVLPs. In some embodiments, the nucleotide sequence encoding M2-1polypeptide comprises the sequence set forth in SEQ ID NO: 44 or SEQ IDNO: 46, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with SEQ ID NO:44 or SEQ ID NO: 46, or a polynucleotide comprising a portion of SEQ IDNO: 44 or SEQ ID NO: 46.

In some embodiments, the VLP of any preceding aspect comprises one ormore additional RSV proteins. In some embodiments, the VLP comprises anRSV F protein. As used herein, the terms “RSV F protein”, “F protein”refers to an RSV fusion protein. The RSV F protein directs penetrationof RSV by fusion between the virion's envelope protein and the host cellplasma membrane. Later in infection, the F protein expressed on the cellsurface can mediate fusion with neighboring cells to form syncytia. TheF protein is a type I transmembrane surface protein that has aN-terminal cleaved signal peptide and a membrane anchor near theC-terminus. RSV F is synthesized as an inactive F0 precursor thatassembles into a homotrimer and is activated by cleavage in thetrans-Golgi complex by a cellular endoprotease to yield twodisulfide-linked subunits. The N-terminus of the F1 subunit that iscreated by cleavage contains a hydrophobic domain (the fusion peptide)that inserts directly into the target membrane to initiate fusion. TheF1 subunit also contains heptad repeats that associate during fusion,driving a conformational shift that brings the viral and cellularmembranes into close proximity. Because the F protein is expressed onthe surface of infected cells and is responsible for subsequent fusionwith other cells leading to syncytia formation, antibodies or cellularimmune responses to the F protein can neutralize virus and/or blockentry of the virus into the cell or prevent syncytia formation.

Accordingly, in some embodiments, the RSV F protein of any precedingaspect is selected from a group consisting of a pre-fusion form of theRSV F protein, a post-fusion form of the RSV F protein, and a carbonylterminal portion of the RSV F protein. In some embodiments, the RSV Fprotein is the pre-fusion form of the RSV F protein. In someembodiments, the RSV F protein is the post-fusion form of the RSV Fprotein. In some embodiments, the RSV F protein is the carbonyl terminalportion of the RSV F protein. In some embodiments, the RSV F proteincomprises a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, and SEQ ID NO: 37.

In some embodiments, the F polypeptide comprises the sequence set forthin SEQ ID NO: 23. In some embodiments, the F polypeptide comprises thesequence set forth in SEQ ID NO: 26. In some embodiments, the Fpolypeptide comprises the sequence set forth in SEQ ID NO: 29. In someembodiments, the F polypeptide comprises the sequence set forth in SEQID NO: 32. In some embodiments, the F polypeptide comprises the sequenceset forth in SEQ ID NO: 34. In some embodiments, the F polypeptidecomprises the sequence set forth in SEQ ID NO: 36. In some embodiments,the F polypeptide comprises the sequence set forth in SEQ ID NO: 37.

The term “RSV F protein” or “F” protein also encompasses nucleotidesequences which encode for an RSV F and/or any variants, derivativesand/or fragments thereof that when transfected (or infected) into a hostcell will express an RSV F protein and induce formation of VLPs. In someembodiments, the RSV F polypeptide comprises the sequence set forth inSEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, or SEQ ID NO: 37, or sequence having at or greaterthan about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%homology with the sequence set forth in SEQ ID NO: 23, SEQ ID NO: 26,SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ IDNO: 37, or a polypeptide comprising a portion of the sequence set forthin SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, thenucleotide sequence encoding the RSV F polypeptide comprises thesequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,or SEQ ID NO: 38, or sequence having at or greater than about 80%, about85%, about 90%, about 95%, about 98%, or about 99% homology with SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 38, or apolynucleotide comprising a portion of SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO: 35, or SEQ ID NO: 38.

In some embodiments, the carbonyl terminal portion form of the RSV Fpolypeptide comprises the sequence set forth in SEQ ID NO: 32, orsequence having at or greater than about 80%, about 85%, about 90%,about 95%, about 98%, or about 99% homology with SEQ ID NO: 32, or apolypeptide comprising a portion of SEQ ID NO: 32. The term alsoencompasses nucleotide sequences which encode for the carbonyl terminalportion form of the RSV F polypeptide and/or any variants, derivativesand/or fragments thereof that when transfected (or infected) into a hostcell will express the carbonyl terminal portion form of the RSV Fpolypeptide and induce formation of VLPs. In some embodiments, thenucleotide sequence encoding the carbonyl terminal portion form of theRSV F polypeptide comprises the sequence set forth in SEQ ID NO: 33, orsequence having at or greater than about 80%, about 85%, about 90%,about 95%, about 98%, or about 99% homology with SEQ ID NO: 33, or apolynucleotide comprising a portion of SEQ ID NO: 33.

In some embodiments, the VLP of any preceding aspect further comprisesan RSV G protein. In some embodiments, the RSV G protein is from RSVgroup A or RSV group B. In some embodiments, the RSV G protein is fromRSV group A. In some embodiments, the RSV G protein is from RSV group B.In some embodiments, the RSV G protein is from RSV group A and RSV groupB.

As used herein, the terms “RSV G protein” or “G protein” refers to atype II transmembrane glycoprotein with a single hydrophobic region nearthe N-terminal end that serves as both an uncleaved signal peptide and amembrane anchor, leaving the C-terminal two-thirds of the moleculeoriented externally. In some embodiments, the RSV G polypeptidecomprises a sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ IDNO: 21, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or apolypeptide comprising a portion of a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, and SEQ ID NO: 21. The term also encompassesnucleotide sequences which encode for the RSV G polypeptide and/or anyvariants, derivatives and/or fragments thereof that when transfected (orinfected) into a host cell will express various forms of the RSV Gprotein and induce formation of VLPs (for example, secreted ormembrane-bound G proteins). In some embodiments, the nucleotide sequenceencoding the RSV G protein comprises a sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, and SEQ ID NO: 22, or sequence having at or greaterthan about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%homology with a sequence selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, andSEQ ID NO: 22, or a polynucleotide comprising a portion of a sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. In someembodiments, the G protein comprises a sequence of SEQ ID NO: 1. In someembodiments, the G protein comprises a sequence of SEQ ID NO: 11. Insome embodiments, the G protein comprises a sequence of SEQ ID NO: 15.In some embodiments, the G protein comprises a sequence of SEQ ID NO:17.

The G protein structure consists of a central conserved region (CCD-G)that contains a CX3C chemokine motif that enables binding to the CX3Cchemokine receptor, CX3CR1 (HGNC: 2558, Entrez Gene: 1524, Ensembl:ENSG00000168329, OMIM: 601470, UniProtKB: P49238), a crucial chemokinereceptor involved in migration and adhesion of leukocytes. The CX3Cmotif is known to those of skill in the art. In some embodiments, theCX3C motif is from amino acid 182 to 186 of an RSV G protein from groupA. G protein induced inflammatory responses can be reduce by blocking Gprotein binding to CX3CR1. The present disclosure shows that the anti-Gprotein antibodies induced by the vaccine disclosed herein and reducesRSV infection related inflammation. Therefore, in some embodiments, theVLPs of any preceding aspect comprises a recombinant RSV G protein,wherein the recombinant RSV G protein comprises an intracellular andtransmembrane domain plus about 20 to about 25 aa of the extracellulardomain and/or a CCD-G of an RSV G protein, wherein the RSV G protein isfrom RSV group A and/or group B. In some embodiments, the transmembranedomain term used herein can indicate intracellular and transmembranedomains plus about 20 to about 25 aa of the extracellular domain (e.g.aa 1-86 or 1-91). In some embodiments, the intracellular plustransmembrane plus initial sequences of the extracellular domain of anRSV G protein comprises amino acids 1-86 of an RSV from group A or aminoacids 1-77 of an RSV from group B. In some embodiments, the CCD-G domainof an RSV G protein comprises amino acid 155-206 of an RSV G protein ofgroup A or B or amino acid 146-197 of an RSV G protein of group A or Bor 146-206 of group A or B. In some embodiments, the RSV G protein ofgroup A comprises a sequence of SEQ ID NO: 7 or SEQ ID NO: 9. In someembodiments, the RSV G protein of group B comprises a sequence of SEQ IDNO: SEQ ID NO: 19 or SEQ ID NO: 21. The CX3C motif can be mutated forpreventing the G protein in the VLP from binding to CX3CR1, such thatthe VLP can be more immunogenic and safer. Therefore, in someembodiments, the CCD-G comprises a mutated CX3C motif that has one ormore amino acids inserted between the two cysteines of the CX3C motif.The one or more amino acids can be any amino acid. In some embodiments,the one or more amino acids are alanines. Accordingly, in someembodiments, the recombinant G protein of any preceding aspect furthercomprises mutated CX3C motif, wherein the recombinant G proteincomprises a sequence selected from the group consisting of SEQ ID NO: 11and SEQ ID NO: 13.

As noted above, the term “engineered” or “recombinant” means apolynucleotide or polypeptide of semisynthetic, or synthetic origin thateither does not occur in nature or is operably linked to anotherpolynucleotide in an arrangement not found in nature. In someembodiments, recombinant RSV G protein is a G protein comprising one ormore transmembrane domains of RSV G proteins and one or more CCD-Gdomains of RSV G proteins, wherein the transmembrane domain and theCCD-G domain can be from a same RSV group or different RSV groups. Insome embodiments, the recombinant RSV G protein comprises atransmembrane domain of an RSV G protein and a CCD-G domain of an RSV Gprotein, wherein the transmembrane domain and the CCD-G domain can befrom a same RSV group or different RSV groups. Thus, in someembodiments, the VLP disclosed herein comprises a recombinant RSV Gprotein comprising a transmembrane domain of an RSV G protein of RSVgroup A and a CCD-G domain of an RSV G protein of RSV group A. In someembodiments, the VLP disclosed herein comprises a recombinant RSV Gprotein comprising a transmembrane domain of an RSV G protein of RSVgroup A and a CCD-G domain of an RSV G protein of RSV group B. In someembodiments, the VLP disclosed herein comprises a recombinant RSV Gprotein comprising a transmembrane domain of an RSV G protein of RSVgroup B and a CCD-G domain of an RSV G protein of RSV group B. In someembodiments, the recombinant RSV G protein comprises a transmembranedomain of an RSV G protein and a group A CCD-G domain and a group BCCD-G domain of RSV G protein, wherein the transmembrane domain can befrom either a group A or B RSV groups. In some embodiments, the VLP canhave two recombinant G proteins, one for group A and one for group B.The recombinant RSV G protein of any preceding aspect, the transmembranedomain is operably linked to the CCD-G domain and the VLP also containsa form of the F protein.

Accordingly, in some embodiments, the recombinant G protein of anypreceding aspect comprises a sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, and SEQ ID NO: 21. In some embodiments, the recombinant G proteincomprises a sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ IDNO: 21, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or apolypeptide comprising a portion of a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, and SEQ ID NO: 21. In some embodiments, thenucleotide sequence encoding the recombinant G protein comprises asequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, orsequence having at or greater than about 80%, about 85%, about 90%,about 95%, about 98%, or about 99% homology with a sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, or a polynucleotidecomprising a portion of a sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, and SEQ ID NO: 22.

In some embodiments, the recombinant G protein comprises a sequence ofSEQ ID NO: 1. In some embodiments, the recombinant G protein comprises asequence of SEQ ID NO: 3. In some embodiments, the recombinant G proteincomprises a sequence of SEQ ID NO: 5. In some embodiments, therecombinant G protein comprises a sequence of SEQ ID NO: 7. In someembodiments, the recombinant G protein comprises a sequence of SEQ IDNO: 9. In some embodiments, the recombinant G protein comprises asequence of SEQ ID NO: 11. In some embodiments, the recombinant Gprotein comprises a sequence of SEQ ID NO: 13. In some embodiments, therecombinant G protein comprises a sequence of SEQ ID NO: 15. In someembodiments, the recombinant G protein comprises a sequence of SEQ IDNO: 17. In some embodiments, the recombinant G protein comprises asequence of SEQ ID NO: 19. In some embodiments, the recombinant Gprotein comprises a sequence of SEQ ID NO: 21.

In some embodiments, the VLP of any preceding aspect further comprisesan RSV P protein, an RSV N protein, or an RSV L protein.

In some aspects, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein, an RSV Pprotein, an RSV F protein, and an RSV G protein.

In some embodiments, the RSV F protein is selected from a groupconsisting of a pre-fusion form of the RSV F protein, a post-fusion formof the RSV F protein, and a carbonyl terminal portion of the RSV Fprotein. In some embodiments, the RSV F protein comprises a sequenceselected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQID NO: 29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:37. In some embodiments, the carbonyl terminal portion of the RSV Fprotein comprises a sequence of SEQ ID NO: 32.

In some embodiments, the VLP comprises an RSV G protein. In someembodiments, the RSV G protein is from RSV group A or RSV group B. Insome embodiments, the RSV G protein comprises a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

In some embodiments, the VLP comprises a recombinant RSV G protein. Insome embodiments, the recombinant RSV G protein comprises atransmembrane domain of the RSV G protein and a central conserved domainof the RSV G protein.

As used herein, the term “RSV P protein” or “P protein” refers to an RSVphosphorylation. The colocalization of M2-1 protein with N protein and Pprotein as part of the ribonucleoprotein (RNP) complex contributes RSVviral gene transcription and replication. An example of an RSV P proteinis represented by SEQ ID NO: 47 or SEQ ID NO: 49. The term alsocomprises any variants, derivatives and/or fragments of RSV P proteinthat, when expressed in a host cell, induces formation of VLPs. In someembodiments, the P polypeptide comprises the sequence set forth in SEQID NO: 47 or SEQ ID NO: 49, or sequence having at or greater than about80%, about 85%, about 90%, about 95%, about 98%, or about 99% homologywith SEQ ID NO: 47 or SEQ ID NO: 49, or a polypeptide comprising aportion of SEQ ID NO: 47 or SEQ ID NO: 49. The term also encompassesnucleotide sequences which encode for RSV P and/or any variants,derivatives and/or fragments thereof that when transfected (or infected)into a host cell will express RSV P protein and induce formation ofVLPs. In some embodiments, the nucleotide sequence encoding RSV Pprotein comprises the sequence set forth in SEQ ID NO: 48 or SEQ ID NO:50, or sequence having at or greater than about 80%, about 85%, about90%, about 95%, about 98%, or about 99% homology with SEQ ID NO: 48 orSEQ ID NO: 50, or a polypeptide comprising a portion of SEQ ID NO: 48 orSEQ ID NO: 50.

In some embodiments, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein, an RSV Pprotein, an RSV F protein, and an RSV G protein, wherein the VLP furthercomprises an RSV M2-1 protein, an RSV N protein, or an RSV L protein.Additional examples of RSV sequences and proteins are further describedin U.S. Patent Application Publication U.S. 2008/0233150, which isincorporated herein by reference for all purposes.

In some aspects, disclosed herein are vaccines comprising VLPs of anypreceding aspect. In some embodiments, the vaccine further comprises anadjuvant.

Optionally, the vaccine contemplated herein can be combined with anadjuvant such as Freund's incomplete adjuvant, Freund's Completeadjuvant, alum, monophosphoryl lipid A, alum phosphate or hydroxide,QS-21, salts, i.e., AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4)2, silica, kaolin,carbon polynucleotides, i.e., poly IC and poly AU. Additional adjuvantscan include QuilA and Alhydrogel and the like. Optionally, the vaccinecontemplated herein can be combined with immunomodulators andimmunostimulants such as interleukins, interferons and the like. Manyvaccine formulations are known to those of skill in the art.

In some embodiments, the vaccine further comprises a pharmaceuticallyacceptable carrier.

METHODS OF USE

In some aspects, disclosed herein is a method of inducing immunity toRSV infection or at least one symptom thereof in a subject, comprisingadministering one or more effective doses of a vaccine comprising avirus like particle (VLP), wherein the VLP comprises a respiratorysyncytial virus (RSV) M protein and an RSV M2-1 protein.

In some embodiments, the VLP further comprises an F protein. In someembodiments, the VLP further comprises a G protein. In some embodiments,the VLP further comprises an F protein and a G protein.

In some aspects, disclosed herein is a method of inducing immunity toRSV infection or at least one symptom thereof in a subject, comprisingadministering one or more effective doses of a vaccine comprising avirus like particle (VLP), wherein the VLP comprises a respiratorysyncytial virus (RSV) M protein, an RSV P protein, an RSV F protein, andan RSV G protein.

In some aspects, disclosed herein is a method of preventing an RSVinfection or at least one symptom thereof in a subject, comprisingadministering one or more effective doses of a vaccine comprising avirus like particle (VLP), wherein the VLP comprises a respiratorysyncytial virus (RSV) M protein and an RSV M2-1 protein. In someembodiments, the VLP further comprises an F protein. In someembodiments, the VLP further comprises a G protein. In some embodiments,the VLP further comprises an F protein and a G protein.

In some aspects, disclosed herein is a method of preventing an RSVinfection or at least one symptom thereof in a subject, comprisingadministering one or more effective doses of a vaccine comprising avirus like particle (VLP), wherein the VLP comprises a respiratorysyncytial virus (RSV) M protein, an RSV P protein, an RSV F protein, andan RSV G protein.

As used herein, the terms “RSV Matrix” or “RSV M” protein refer to anRSV protein that, when expressed in a host cell, induces formation ofVLPs. An example of an RSV M protein is represented by SEQ ID NO: 39 orSEQ ID NO: 41. The term also comprises any variants, derivatives and/orfragments of RSV M protein that, when expressed in a host cell, inducesformation of VLPs. In some embodiments, the M polypeptide comprises thesequence set forth in SEQ ID NO: 39 or SEQ ID NO: 41, or sequence havingat or greater than about 80%, about 85%, about 90%, about 95%, about98%, or about 99% homology with SEQ ID NO: 39 or SEQ ID NO: 41, or apolypeptide comprising a portion of SEQ ID NO: 39 or SEQ ID NO: 41. Theterm also encompasses nucleotide sequences which encode for RSV M and/orany variants, derivatives and/or fragments thereof that when transfected(or infected) into a host cell will express RSV M protein and induceformation of VLPs. In some embodiments, the nucleotide sequence encodingM polypeptide comprises the sequence set forth in SEQ ID NO: 40 or SEQID NO: 42, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with SEQ ID NO:40 or SEQ ID NO: 42, or a polynucleotide comprising a portion of SEQ IDNO: 40 or SEQ ID NO: 42.

As used herein, the terms “RSV M2-1 protein”, “M2-1 protein” refers to acofactor of the RSV viral RNA polymerase complex and functions as atranscriptional processivity and antitermination factor. An example ofan RSV M2-1 protein is represented by SEQ ID NO: 43 or SEQ ID NO: 45.The term also comprises any variants, derivatives and/or fragments ofRSV M2-1 protein that, when expressed in a host cell, induces formationof VLPs. In some embodiments, the M2-1 polypeptide comprises thesequence set forth in SEQ ID NO: 43 or SEQ ID NO: 45, or sequence havingat or greater than about 80%, about 85%, about 90%, about 95%, about98%, or about 99% homology with SEQ ID NO: 43 or SEQ ID NO: 45, or apolypeptide comprising a portion of SEQ ID NO: 43 or SEQ ID NO: 45. Theterm also encompasses nucleotide sequences which encode for RSV M2-1and/or any variants, derivatives and/or fragments thereof that whentransfected (or infected) into a host cell will express RSV M2-1 proteinand induce formation of VLPs. In some embodiments, the nucleotidesequence encoding M2-1 polypeptide comprises the sequence set forth inSEQ ID NO: 44 or SEQ ID NO: 46, or sequence having at or greater thanabout 80%, about 85%, about 90%, about 95%, about 98%, or about 99%homology with SEQ ID NO: 44 or SEQ ID NO: 46, or a polynucleotidecomprising a portion of SEQ ID NO: 44 or SEQ ID NO: 46.

In some embodiments, the VLP of any preceding aspect comprises one ormore additional RSV proteins. In some embodiments, the VLP comprises anRSV F protein. As used herein, the terms “RSV F protein”, “F protein”refers to an RSV fusion protein.

Accordingly, in some embodiments, the RSV F protein of any precedingaspect is selected from a group consisting of a pre-fusion form of theRSV F protein, a post-fusion form of the RSV F protein, and a carbonylterminal portion of the RSV F protein. In some embodiments, the RSV Fprotein is the pre-fusion form of the RSV F protein. In someembodiments, the RSV F protein is the post-fusion form of the RSV Fprotein. In some embodiments, the RSV F protein is the carbonyl terminalportion of the RSV F protein. Accordingly, in some embodiments, the RSVF protein of any preceding aspect is selected from a group consisting ofa pre-fusion form of the RSV F protein, a post-fusion form of the RSV Fprotein, and a carbonyl terminal portion of the RSV F protein. In someembodiments, the RSV F protein is the pre-fusion form of the RSV Fprotein. In some embodiments, the RSV F protein is the post-fusion formof the RSV F protein. In some embodiments, the RSV F protein is thecarbonyl terminal portion of the RSV F protein. In some embodiments, theRSV F protein comprises a sequence selected from the group consisting ofSEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, and SEQ ID NO: 37.

The term “RSV F protein” or “F” protein also encompasses nucleotidesequences which encode for an RSV F and/or any variants, derivativesand/or fragments thereof that when transfected (or infected) into a hostcell will express an RSV F protein and induce formation of VLPs. In someembodiments, the RSV F polypeptide comprises the sequence set forth inSEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, or SEQ ID NO: 37, or sequence having at or greaterthan about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%homology with the sequence set forth in SEQ ID NO: 23, SEQ ID NO: 26,SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ IDNO: 37, or a polypeptide comprising a portion of the sequence set forthin SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, or SEQ ID NO: 37. In some embodiments, thenucleotide sequence encoding the RSV F polypeptide comprises thesequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,or SEQ ID NO: 38, or sequence having at or greater than about 80%, about85%, about 90%, about 95%, about 98%, or about 99% homology with SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 38, or apolynucleotide comprising a portion of SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO: 35, or SEQ ID NO: 38.

In some embodiments, the carbonyl terminal portion form of the RSV Fpolypeptide comprises the sequence set forth in SEQ ID NO: 32, orsequence having at or greater than about 80%, about 85%, about 90%,about 95%, about 98%, or about 99% homology with SEQ ID NO: 32, or apolypeptide comprising a portion of SEQ ID NO: 32. The term alsoencompasses nucleotide sequences which encode for the carbonyl terminalportion form of the RSV F polypeptide and/or any variants, derivativesand/or fragments thereof that when transfected (or infected) into a hostcell will express the carbonyl terminal portion form of the RSV Fpolypeptide and induce formation of VLPs. In some embodiments, thenucleotide sequence encoding the carbonyl terminal portion form of theRSV F polypeptide comprises the sequence set forth in SEQ ID NO: 33, orsequence having at or greater than about 80%, about 85%, about 90%,about 95%, about 98%, or about 99% homology with SEQ ID NO: 33, or apolynucleotide comprising a portion of SEQ ID NO: 33.

In some embodiments, the VLP of any preceding aspect further comprisesan RSV G protein. In some embodiments, the RSV G protein is from RSVgroup A or RSV group B. In some embodiments, the RSV G protein is fromRSV group A. In some embodiments, the RSV G protein is from RSV group B.In some embodiments, the RSV G protein is from RSV group A and RSV groupB.

As used herein, the terms “RSV G protein” or “G protein” refers to atype II transmembrane glycoprotein with a single hydrophobic region nearthe N-terminal end that serves as both an uncleaved signal peptide and amembrane anchor, leaving the C-terminal two-thirds of the moleculeoriented externally. In some embodiments, the RSV G polypeptidecomprises a sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ IDNO: 21, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or apolypeptide comprising a portion of a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, and SEQ ID NO: 21. The term also encompassesnucleotide sequences which encode for the RSV G polypeptide and/or anyvariants, derivatives and/or fragments thereof that when transfected (orinfected) into a host cell will express the post-fusion form of the RSVG and induce formation of VLPs. In some embodiments, the nucleotidesequence encoding the RSV G protein comprises a sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, or sequence having at orgreater than about 80%, about 85%, about 90%, about 95%, about 98%, orabout 99% homology with a sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, and SEQ ID NO: 22, or a polynucleotide comprising a portion of asequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. Insome embodiments, the G protein comprises a sequence of SEQ ID NO: 1. Insome embodiments, the G protein comprises a sequence of SEQ ID NO: 11.In some embodiments, the G protein comprises a sequence of SEQ ID NO:15. In some embodiments, the G protein comprises a sequence of SEQ IDNO: 17.

The G protein structure consists of a central conserved region (CCD-G)that contains a CX3C chemokine motif that enables binding to the CX3Cchemokine receptor, CX3CR1 (HGNC: 2558, Entrez Gene: 1524, Ensembl:ENSG00000168329, OMIM: 601470, UniProtKB: P49238), a crucial chemokinereceptor involved in migration and adhesion of leukocytes. G proteininduced inflammatory responses can be reduce by blocking G proteinbinding to CX3CR1. The present disclosure shows that the anti-G proteinantibodies induced by the vaccine disclosed herein reduce RSV infectionrelated inflammation. Therefore, in some embodiments, the VLPs of anypreceding aspect comprises a recombinant RSV G protein, wherein therecombinant RSV G protein comprises a transmembrane domain of an RSV Gprotein and/or a CCD-G of an RSV G protein, wherein the RSV G protein isfrom RSV group A and/or group B. In some embodiments, the transmembranedomain of an RSV G protein comprises amino acid 1-86 of an RSV fromgroup A, amino acid 1-86 of an RSV from group B, or amino acid 1-77 ofan RSV from group B. In some embodiments, the CCD-G domain of an RSV Gprotein comprises amino acid 155-206 of an RSV G protein of group A,amino acid 155-206 of an RSV G protein of group B, or amino acid 146-197of an RSV G protein of group B. In some embodiments, the RSV G proteinof group A comprises a sequence of SEQ ID NO: 7 or SEQ ID NO: 9. In someembodiments, the RSV G protein of group B comprises a sequence of SEQ IDNO: SEQ ID NO: 19 or SEQ ID NO: 21.

In some embodiments, recombinant RSV G protein is a G protein comprisingone or more transmembrane domains of RSV G proteins and one or moreCCD-G domains of RSV G proteins, wherein the transmembrane domain andthe CCD-G domain can be from a same RSV group or different RSV groups.In some embodiments, the recombinant RSV G protein comprises atransmembrane domain of an RSV G protein and a CCD-G domain of an RSV Gprotein, wherein the transmembrane domain and the CCD-G domain can befrom a same RSV group or different RSV groups. Thus, in someembodiments, the VLP disclosed herein comprises a recombinant RSV Gprotein comprising a transmembrane domain of an RSV G protein of RSVgroup A and a CCD-G domain of an RSV G protein of RSV group A. In someembodiments, the VLP disclosed herein comprises a recombinant RSV Gprotein comprising a transmembrane domain of an RSV G protein of RSVgroup A and a CCD-G domain of an RSV G protein of RSV group B. In someembodiments, the VLP disclosed herein comprises a recombinant RSV Gprotein comprising a transmembrane domain of an RSV G protein of RSVgroup B and a CCD-G domain of an RSV G protein of RSV group B. In someembodiments, the recombinant RSV G protein comprises more than onetransmembrane domain of an RSV G protein and more than one CCD-G domainof an RSV G protein, wherein the transmembrane domain and the CCD-Gdomain can be from a same RSV group or different RSV groups, wherein theRSV group can be RSV group A and/or group B. The recombinant RSV Gprotein of any preceding aspect, the transmembrane domain is operablylinked to the CCD-G domain.

Accordingly, in some embodiments, the recombinant G protein of anypreceding aspect comprises a sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, and SEQ ID NO: 21. In some embodiments, the recombinant G proteincomprises a sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ IDNO: 21, or sequence having at or greater than about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% homology with a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or apolypeptide comprising a portion of a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, and SEQ ID NO: 21. In some embodiments, thenucleotide sequence encoding the recombinant G protein comprises asequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, orsequence having at or greater than about 80%, about 85%, about 90%,about 95%, about 98%, or about 99% homology with a sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, or a polynucleotidecomprising a portion of a sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, and SEQ ID NO: 22. In some embodiments, the recombinant G proteincomprises a sequence of SEQ ID NO: 1. In some embodiments, therecombinant G protein comprises a sequence of SEQ ID NO: 3. In someembodiments, the recombinant G protein comprises a sequence of SEQ IDNO: 5. In some embodiments, the recombinant G protein comprises asequence of SEQ ID NO: 7. In some embodiments, the recombinant G proteincomprises a sequence of SEQ ID NO: 9. In some embodiments, therecombinant G protein comprises a sequence of SEQ ID NO: 11. In someembodiments, the recombinant G protein comprises a sequence of SEQ IDNO: 13. In some embodiments, the recombinant G protein comprises asequence of SEQ ID NO: 15. In some embodiments, the recombinant Gprotein comprises a sequence of SEQ ID NO: 17. In some embodiments, therecombinant G protein comprises a sequence of SEQ ID NO: 19. In someembodiments, the recombinant G protein comprises a sequence of SEQ IDNO: 21.

In some embodiments, the VLP of any preceding aspect further comprisesan RSV P protein, an RSV N protein, or an RSV L protein.

In some aspects, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein, an RSV Pprotein, an RSV F protein, and an RSV G protein.

In some embodiments, the RSV F protein is selected from a groupconsisting of a pre-fusion form of the RSV F protein, a post-fusion formof the RSV F protein, and a carbonyl terminal portion of the RSV Fprotein. In some embodiments, the RSV F protein comprises a sequenceselected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQID NO: 29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:37. In some embodiments, the carbonyl terminal portion of the RSV Fprotein comprises a sequence of SEQ ID NO: 32.

In some embodiments, the VLP comprises an RSV G protein. In someembodiments, the RSV G protein is from RSV group A or RSV group B. Insome embodiments, the RSV G protein comprises a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

As used herein, the terms “RSV P protein”, “P protein” refers to an RSVphosphorylation. The colocalization of M2-1 protein with N protein and Pprotein as part of the ribonucleoprotein (RNP) complex contributes RSVviral gene transcription and replication. An example of an RSV P proteinis represented by SEQ ID NO: 47 or SEQ ID NO: 49. The term alsocomprises any variants, derivatives and/or fragments of RSV P proteinthat, when expressed in a host cell, induces formation of VLPs. In someembodiments, the P polypeptide comprises the sequence set forth in SEQID NO: 47 or SEQ ID NO: 49, or sequence having at or greater than about80%, about 85%, about 90%, about 95%, about 98%, or about 99% homologywith SEQ ID NO: 47 or SEQ ID NO: 49, or a polypeptide comprising aportion of SEQ ID NO: 47 or SEQ ID NO: 49. The term also encompassesnucleotide sequences which encode for RSV P and/or any variants,derivatives and/or fragments thereof that when transfected (or infected)into a host cell will express RSV P protein and induce formation ofVLPs. In some embodiments, the nucleotide sequence encoding RSV Pprotein comprises the sequence set forth in SEQ ID NO: 48 or SEQ ID NO:50, or sequence having at or greater than about 80%, about 85%, about90%, about 95%, about 98%, or about 99% homology with SEQ ID NO: 48 orSEQ ID NO: 50, or a polypeptide comprising a portion of SEQ ID NO: 48 orSEQ ID NO: 50.

In some embodiments, disclosed herein is a virus like particle (VLP)comprising a respiratory syncytial virus (RSV) M protein, an RSV Pprotein, an RSV F protein, and an RSV G protein, wherein the VLP furthercomprises an RSV M2-1 protein, an RSV N protein, or an RSV L protein.

In some aspects, disclosed herein are vaccines comprising VLPs of anypreceding aspect. In some embodiments, the vaccine further comprises anadjuvant.

Optionally, the vaccine contemplated herein can be combined with anadjuvant such as Freund's incomplete adjuvant, Freund's Completeadjuvant, alum, monophosphoryl lipid A, alum phosphate or hydroxide,QS-21, salts, i.e., AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4)2, silica, kaolin,carbon polynucleotides, i.e., poly IC and poly AU. Additional adjuvantscan include QuilA and Alhydrogel and the like. Optionally, the vaccinecontemplated herein can be combined with immunomodulators andimmunostimulants such as interleukins, interferons and the like. Manyvaccine formulations are known to those of skill in the art.

In some embodiments, the vaccine further comprises a pharmaceuticallyacceptable carrier.

The vaccines of the present invention can be administered to theappropriate subject in any manner known in the art, e.g., orallyintramuscularly, intravenously, sublingual mucosal, intraarterially,intrathecally, intradermally, intraperitoneally, intranasally,intrapulmonarily, intraocularly, intravaginally, intrarectally orsubcutaneously. They can be introduced into the gastrointestinal tractor the respiratory tract, e.g., by inhalation of a solution or powdercontaining the conjugates. In some embodiments, the compositions can beadministered via absorption via a skin patch. Parenteral administration,if used, is generally characterized by injection. Injectables can beprepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system, such that a constant level of dosage ismaintained. In some embodiments, the one or more effective doses of thevaccine are administered to the subject via a route that is selectedfrom the group consisting of an intramuscular route, a subcutaneousroute, an intradermal route, an oral administration, a nasaladministration, and inhalation.

A pharmaceutical composition (e.g., a vaccine) is administered in anamount sufficient to elicit production of antibodies and activation ofCD4+ T cells and CD8+ T cells as part of an immunogenic response. Dosagefor any given patient depends upon many factors, including the patient'ssize, general health, sex, body surface area, age, the particularcompound to be administered, time and route of administration, and otherdrugs being administered concurrently. Determination of optimal dosageis well within the abilities of a pharmacologist of ordinary skill.

The vaccine compositions are administered to subjects which may becomeinfected by a Listeria described herein, including but not limited todogs, cats, rabbits, rodents, horses, livestock (e.g., cattle, sheep,goats, and pigs), zoo animals, ungulates, primates, and humans. In someembodiments, the preferred subject is a human. In some embodiments, thesubject is an infant or a child. In some embodiments, the human has anage less than 15 years old, 12 years old, 11 years old, 10 years old, 9years old, 8 years old, 7 years old, 6 years old, 5 years old, 4 yearsold, 3 years old, 2 years old, or 1 year old.

EXAMPLES

The following examples are set forth below to illustrate the compounds,systems, methods, and results according to the disclosed subject matter.These examples are not intended to be inclusive of all aspects of thesubject matter disclosed herein, but rather to illustrate representativemethods and results. These examples are not intended to excludeequivalents and variations of the present invention which are apparentto one skilled in the art.

Example 1. Expression of G and F on RSV M Based Virus-Like Particles

VLPs were generated from sequentially transfecting 293F cells withcodon-optimized DNA plasmids containing RSV genes. The order oftransfection is listed in Table 1. Western blot studies showed thesuccessful development of VLPs with the M plus phosphoprotein P or the Mplus M2-1 protein platforms with F or F and G (FIG. 1A), but only tovery low levels with G alone, a condition not observed in VLPsexpressing only F (FIG. 1C). The Western blot studies show detection ofF or F and G in the supernatant and not the cell pellet of the induced293F cells. Additionally, F or F and G were detected by Western blot inthe pellet after centrifugation through a sucrose cushion indicating theproteins were particles and not dissolved in the media. Finally, F and Mor F, G, and M were detected by Western blot in the same fractions aftersucrose gradient purification indicating successful expression andpurified of VLPs containing F or F and G (FIGS. 1A and 1B). The factthat G alone led to very low expression in VLPs and G with F gave highlevels of VLPs with both indicated that F facilitated incorporation of Ginto VLPs. To further test this, a truncated F that consisted of thecarbonyl terminus+transmembrane+26 amino acids of the amino terminus(F_(t)) was generated and this peptide was co-expressed with full-lengthG on M+P flatform. The data show that in the presence of F_(t), Gexpression was abundant (FIG. 1C).

TABLE 1 Generation of RSV VLPs Order of transfection VLPs 1^(st) 2^(nd)3^(rd) 4^(th) M M MFP M P F MFPG M P F G MFPG_(P) M P F G_(P) MFM2-1 MM2-1 F MFM2-1G M M2-1 F G MFM2-1G_(P) M M2-1 F G_(P)

Since this platform allows incorporating different F and G constructsinto the VLPs, further modification was done to make the F proteinconstructs most effective at inducing an anti-viral response and Gprotein constructs most effective at inducing anti-viral,anti-inflammatory responses. For example, RSV VLPs with M, M2-1, F and atruncated G (referred to as “G_(P)”; intracellular, transmembrane, andthe 1^(st) 20 aa of the extracellular domain plus aa 155-206 of the Gprotein) were developed. This construct focuses on inducing antibodiesagainst CCD-G, not other parts of G, and improves anti-CCD-G antibodytiters. Because G's central conserved domain is crucial for the vaccinesdeveloped herein, the current study generated a G peptide that consistedof amino acids 1-86+155-206 (G_(P)) and generated full-length F and thisG_(P) on the same VLPs utilizing either M+P or M+M2-1 flatform.Immunoblotting results from sucrose gradient show F, G_(P), and M bandsfrom the same fractions, indicating that they were from the sameparticles (FIG. 1B).

RSV M protein based VLPs with M2-1+F, M2-1+F+G, and P+F+G have not beenreported previously. The M2-1 protein has two advantages over the Pprotein; it is part of the structural proteins that stabilize the RSVvirion structure, i.e. better models the natural virus, and itfacilitates T-cells immune responses, making RSV M+M2-1 VLPs an idealRSV vaccine. Importantly, with either the RSV M+P or M+M2-1 VLPs, thestructure of G can be modified to focus the response on the part of Gimportant for inducing anti-inflammatory and anti-viral immuneresponses, i.e. the central conserved domain of G (CCD-G). CCD-Gincludes amino acids 155-206 on the G protein. The sequences are highlyconserved within but not between the two antigenic/genetic groups ofRSV, A and B. In primary human airway epithelial cells and in humaninfection, CX3CR1 is an important receptor for infection and anti-CCD-Gantibodies have an added anti-viral effect in humans.

Example 2. Electron Microscopic Study of VLPs

Electron microscopic studies of both MGFP and MFGM2-1 were performed toconfirm the expression of G and F on VLPs. Negative stain showing spikeson VLP surface which were confirmed to include F spikes and/or G spikesby immunogold labeling with human anti-F motavizumab or human anti-G 3D3monoclonal antibodies. Thus, the above data confirm successfulgeneration of RSV matrix protein M+P or M+M2-1 based VLPs expressingboth G and F.

Example 3. Immunogenicity of VLPs

To determine the immunogenicity of the VLPs, BALB/c mice (n=4) wereimmunized with various VLPs as detailed in Table 2. All immunizedanimals were challenged with 10⁶ TCID50 of RSV r19F at 4 weeks after the2^(n), booster, immunization (FIG. 3A). Blood specimens were collectedbefore challenge and tested for F, Ga, and Gb binding antibodies by EIAand neutralizing antibodies by a micro-neutralization assay. All F and GVLP immunized animals and none of the control M VLP immunized animalsdeveloped F and Ga antibodies. Only one animal produced antibodiesagainst Gb antigen (FIG. 3B). In addition, the short G peptide G_(P) wasmore efficient at inducing antibody than its full-length counterpart,indicating that the central conserved domain of G is an effectiveimmunogen. Furthermore, VLP antigens that contained P induced antibodiesagainst F better than those that contained M2-1 (FIG. 3B). In thisstudy, the VLPs induced low levels of neutralizing antibodies. As shown,sera from animals in groups immunized with MFP and MFM2-1G_(P) did notpossess neutralizing activity, but sera from other groups all had someneutralizing activity (FIG. 3C).

TABLE 2 Immunization schedule RSV Dose Immuni- challenge (per zationTCID50/ Days of Group N mouse) days Route mouse harvest M 4 50 μg VLPs0, 21 IM 10⁶ at 54 day 49 MFP ″ ″ ″ ″ 10⁶ at ″ day 49 MFGP ″ ″ ″ ″ 10⁶at ″ day 49 MFG_(P)P ″ ″ ″ ″ 10⁶ at ″ day 49 MFM2-1 ″ ″ ″ ″ 10⁶ at ″ day49 MFGM2-1 ″ ″ ″ ″ 10⁶ at ″ day 49 MFG_(P)M2-1 ″ ″ ″ ″ 10⁶ at ″ day 49

Example 4. Immunized Animals have Reduced Lung Viral Titers

Next, the ability of the VLPs to prevent virus replication afterchallenge was investigated. The relative cycle threshold (CT) valueswere significant higher indicating less virus replication in animalgroups that had P protein as part of the antigen VLPs, i.e. MFP, MFGP,and MFG_(P)P compared to the control antigen (M only VLPs). A highervalue of CT correlates with low copy of the gene being evaluated. Also,there were significant differences between MFG_(P)P and MFP or MFGP(FIG. 4), indicating that anti-G antibodies participate in viralclearance in the lungs and that G_(P) was more effective in facilitatingvirus clearance than full length G. Moreover, VLP antigens thatcontained M2-1 protein, i.e. MFM2-1, MFGM2-1, and MFG_(P)M2-1 had higherCT values compared to control (FIG. 4).

Example 5. Lung histopathology

Since one of the manifestations of RSV infection with r19F virus isoverproduction of mucin, pulmonary inflammation was examined inchallenged animals by Periodic acid-Schiff staining (PAS). The stainedslides were analyzed by Aperio ImageScope software (Leica, Germany) andscored blindly using 0-4 severity scale then converted to 0-100histopathology scale. Positive PAS staining was found, indicating thepresence of mucin, in the lungs of all immunized animals but less sothan the control animals immunized with M only VLPs (FIGS. 5A and 5B).Only MFG_(P)P- and MFG_(P)M2-1-immunized animals had a significantreduction of PAS staining compared to control animals though (FIGS. 5Aand 5B). These data show that F plus Gp VLPs were most effective atreducing both lung virus replication and mucin production.

Example 6. Discussion

An RSV based VLP platform is developed herein to express F+G, F+Gpeptide, or G plus a truncated F. The G plus truncated F VLP makes itpossible to study G without most of the extracellular F protein, andapproximates the structure, function, and immunogenicity of G in an RSVVLP platform without F. The carboxy terminal intracellular plustransmembrane plus a part of the initial extracellular domain of F wasneeded to efficiently incorporate G into the VLPs. These data show thatthe RSV VLP platform has advantages over the platforms previously usedin Newcastle disease or influenza virus or bacterial phage P22 platformswith the RSV F and/or G proteins.

The RSV platform VLPs better represent native structures of F and G, andfacilitate studies of their structure and function relationships. Italso provides an all RSV antigen platform to develop F and G VLPvaccines. A truncated F (F_(t)) that consists of the carbonylterminus+transmembrane+26 amino acids of the amino terminus wasgenerated and was co-expressed with a full-length G in M+P RSV VLPplatform. This led to efficient G+Ft VLPs which can be used to study Gwith minimal interference by extracellular F. The data also determinedthat peptides of extracellular G can be incorporated as a mean to focusresponses to certain regions of G. Studies show a critical role of thecentral conserved domain of G (CCD-G) in RSV disease pathogenesis andgenerating anti-CCD-G antibodies decreases disease through both ananti-viral and anti-inflammatory effect. CCD-G contains the CX3C motifthrough which G binds to CX3CR1 and antibodies that block binding toCX3CR1 decreases infection since CX3CR1 is an important receptor forinfection of human airway epithelial cells. Studies in animals and inhuman cells in vitro show that blocking binding to CX3CR1 effects theinflammatory response to RSV. The present disclosure shows thesuccessful generation of VLPs with a truncated G composed of amino acids1-86+155-206 of G (G_(P)) with F in both M+P and M+M2-1 platforms.

P plays a crucial role in RSV polymerase activity interacting with bothN and L protein in the process and also interacts with M2-1, but theseroles do not suggest how it might help VLP formation when co-expressedwith M. The M2-1 protein has transcription anti-termination activity anddirectly interacts with the M matrix protein providing a link to the RNAcontaining nucleocapsid.

Electron microscope negative stain studies revealed that both MFGP andMFGM2-1 VLPs expressed glycoproteins F and G as “spikes”. Immunoelectronmicroscopic studies confirmed the presence of F and G on VLP surfaces.No significant electron microscopic differences were observed betweenthe two platforms of VLPs. The mouse studies show that the VLPs wereimmunogenic and induced serum antibodies against both F and G proteinsat similar levels with either platform. Notably, the VLPs with Gpinduced higher antibody titers and were more effective than VLPs withfull length G. The M2-1 protein is effective at inducing short termprotective T cell immunity, making it an ideal antigen for vaccinedesign. Of note, the G protein sequences used in this study are from agroup A strain, A2 and these G sequences efficiently induced antibodiesagainst a group A but not a group B G protein. Note that though CCD-Gregion, with the exception of a 13 amino acid sequence, is distinctbetween group A and group B strains, most of CCD-G is conserved within agroup. Also, serum neutralizing antibody titers were tested, showingthat immunized animals developed low levels of neutralizing antibodies.VLPs with a pre-fusion stabilized F protein can be more effective atinducing neutralizing antibodies. Furthermore, animals immunized withG_(P) and F expressed in either M plus P or M plus M2-1 VLPs had reducedlung viral titer and lung inflammation compared to those immunized withthe control VLPs, demonstrating that both VLP platforms were effective.

The current VLPs described herein can express F and/or G with two RSVplatforms (M+P proteins or M+M2-1 proteins). With both platforms Fand/or G can be modified to focus specific structural and antigenicfeatures of the proteins. Given the failures in RSV vaccine development,combining the highly effective anti-viral activity of F-inducedantibodies plus anti-inflammatory and immune-enhancing features of Gprovides an effective RSV vaccine.

Example 7. Materials and Methods

Cells, Media and Plasmids: 293F cells stably transfected with plasmidpcDNA6/TR were provide by Dr. Xuemin Chen (Emory University) andcultured in freestyle 293 media (Gibco) on a shaker at 37° C., 8% CO₂.pcDNA3.1 DNA plasmids containing codon-optimized RSV genes M, M2-1, P, Gfrom A2 strain, and F from A2 strain were provided by Dr. Marty Moore(now at Meissa Vaccines) were digested by KpnI and XhOI enzymes andcloned into KpnI and XhOI double digested pcDNA4/TO or pcDNA5/TO vector.Human codon optimized truncated Gof amino acids 1-86+155-206 andsynthesized by Genescript (Piscataway, N.J.). The gene provided in pUC57plasmid was double digested by BamHI and XhOI enzymes and cloned intoBamHI and XhOI double digested pcDNA5/TO vector. All genes weresequenced to confirm authenticity prior to transfection. To generateVLPs, 293F cells were sequentially and stably transfected with the RSVgenes noted below.

Virus-like particles expression and purification: 20-30×10⁶ 293F cellswere induced with 2 μg/ml of doxycycline for 72 h. Cells werecentrifuged at 300×g for 10 min and the VLP-containing media wasfiltered through 0.45 μm filter followed by centrifugation through 20%sucrose cushion at 12,200×g for 2 h at 4° C. (SW Ti 32 rotor, OptimaL-90K Ultracentrifuge, Beckman Coulter). The top layer of cell media andsucrose was thoroughly removed and the pellet was soaked in sterile PBSfor 1 h on ice and resuspended. For sucrose gradient experiments,preparation of a linear sucrose gradient was described previously [StoneA B et al., 2009], 1 ml of the gradient was removed before theresuspended VLPs was layered onto the gradient and centrifuged with aBeckman Coulter SW 41 rotor at 11,000×g for 12 h at 4° C. A total of 101-ml fractions were removed from top, diluted 3× with sterile PBS, andcentrifuged at 12,000×g for 1 h at 4° C. on a bench-top refrigerator.Supernatants were completely removed and pellets were soaked in sterilePBS for 1 h on ice before being resuspended.

Antibodies and Immunoblotting: the anti-G protein monoclonal antibody(mAb) 3D3 was provide by Trellis Bioscience (Redwood City, Calif.); theanti-F protein mAb motavizumab was provided by MedImmune (Gaithersburg,Md.); rabbit serum anti-M antibody was provided by Dr. Oomens (OklahomaState University); and goat anti RSV antibody was obtained fromMillipore (Burlington, Mass.). All anti-species fluorescent-conjugatedsecondary antibodies used in immunoblotting were obtained from LI-CORbiosciences (Lincoln, Nebr.). All HRP-conjugated secondary antibodiesused in enzyme-linked immunosorbent assays (EIAs) were obtained fromJackson ImmunoResearch (West Grove, Pa.). For immunoblottingexperiments, VLP samples were mixed with 2× Laemmli sample buffer(Bio-Rad) and boiled at 95° C. for 5 min. Samples were run on SDS-PAGE,transferred to a nitrocellulose membrane, blocked for 30 min in blockingbuffer (5% dry milk in TTBS). After blotting with primary antibody(incubation period), membrane was washed 3× in TTBS following secondaryantibody incubation (time) and 3× washes in TTBS. Signals werevisualized by Odyssey CLX imaging system (LI-COR).

F, Ga, and Gb antibody EIAs: the secreted form of F, Ga or Gb proteinantigens was produced from stably transfected 293F cells in serum-freemedia and coated onto 96-well microtiter plate in buffer. The plateswere incubated in 2% nonfat dry milk dissolved in PBS blocking solutionfor 2 h at 37° C., washed with PBS-T, and serum specimens at 1:200dilution added to the wells, incubated for 1 h at 37° C., the plateswashed with PBS-T, and goat anti mouse IgG-HRP (1:5,000) added andincubated for 1 h at 37° C. Color was developed with OPD substrate andthe reaction stopped after r 30 min at RT with 4N H2SO4. Optical density(OD) was measured at 490 nm and geometric mean of the OD₄₉₀ wascalculated from the triplicate wells.

RSV neutralizing antibody assay: heat inactivated sera were serially2-fold diluted starting with a 1:10 dilution in MEM containing 0.5% FBS,incubated with RSV/A2 (100 TCID50) for 1 h at RT, and inoculated intriplicates onto non-confluent HEp-2 monolayers in 96-well plates for 1h at 37° C. in a 5% CO₂ incubator. MEM containing 5% FBS was added toall the wells and cells were incubated for 3 days at 37° C. in a 5% CO₂incubator. Cells were washed with PBS and fixed with 4% paraformaldehydeand RSV antigens detected by EIA with goat anti RSV antibody (1:5,000)followed with donkey anti goat IgG-HRP secondary antibody (1:5,000).Color was developed with OPD substrate and neutralization defined as a≥50% reduction in OD value. The titer was estimated using the Reed andMuch method. The geometric means±SEM for all animals in a group at anygiven time were calculated.

Virus: A recombinant virus of RSV A2 backbone expressing the F proteinfrom L19 virus (r19F) [Moore M L et al., 2009] was chosen as thechallenge virus since it induces airway disease that parallel RSVinfection in humans but not seen with RSV A2. Stock virus was preparedby inoculating onto subconfluent HEp-2 at a multiplicity of infection(MOI) 0.01 for 2 h at 37° C. in 5% CO₂ incubator using 0.5% fetal bovineserum (FBS)-containing minimal essential medium (MEM). 5% FBS MEM wasadded and cells were incubated in 37° C., 5% CO₂ incubator for 3 days.Cells were frozen and thawed twice at −80 C and 4° C., respectively, andcentrifuged at 2,000 rpm for 15 min at 4° C. to remove cellular debris.The supernatant was layered onto 20% sucrose layer and centrifuged at12,200×g for 2 h at 4° C. Pellet was resuspended in serum free MEM,divided into aliquots, and snap frozen in liquid nitrogen. The aliquotswere stored at −80° C. until use. The infectivity titer of the inoculumwas determined by serial 10-fold dilutions in subconfluent HEp-2 cellmonolayer for 3 days and virus replication detected by EIA for RSVantigens with goat-anti RSV antibody. Titer was estimated from wellswith absorbance >3 standard deviations above the mean absorbance forwells without virus by Reed and Muench method.

Animal study: 4-6 weeks old female BALB/c mice were purchased fromCharles River Lab (Wilmington, Mass.) and housed at Emory's Pediatricanimal facility under food ad libitum in microisolator cages with autosterilized water. All animal handlings and procedures were carried outaccording to protocol approved by Emory University (Atlanta, Ga.)Institutional Animals Care and Use Committee. For challenge study, micewere intranasally infected with 10⁶ CTID50 RSV r19F A2 in 40 μl.

Real-time PCR: total RNA was extracted and purified from lunghomogenates using Qiagen RNeasy kit (QIAGEN). RNA was reversetranscribed into cDNA using iScript™ cDNA synthesis kit (Bio-Rad)following the manufacturer's instruction. Quantitative PCR was carriedout on a 7500 Fast Real-time PCR system (Applied Biosystems) using PowerSYBR Green PCR master mix (Applied Biosystems). CT values werenormalized using control j-actin CT values from the same samples. RSVmatrix M gene primers and amplification cycles were described previously[Boyoglu-Barnum S et al., 2017]. Other primer pairs used were: β-actin,forward 5′-CAC CAA CTG GGA CGA CAT-3′, reverse 5′-ACA GCC TGG ATA GCAACG-3′. mRNA levels were expressed as the geometric mean±SEM for allanimals in a group.

Pulmonary histopathology: lungs were isolated and fixed in 10% neutralbuffered formalin for 24 h. The lungs were then embedded in paraffin,sectioned, and stained with Periodic acid-Schiff (PAS). Slides wereanalyzed by Aperio ImageScope software (Leica) and scored blinded totreatment on a 0-4 scale and subsequently converted to a 0-100%histopathology scale.

Statistical Analysis: unless otherwise indicated, different groups werecompared by Wilcoxon rank sum test or Wilcoxon matched pairs test. A pvalue of <0.05 was considered statistically significant. Data are shownas means and standard errors of the mean (SEM).

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Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

SEQUENCES SEQ ID NO: 1(amino acid sequence for RSV G truncated CX3C aa 1-86 + 155-206 codon-optimized (A strain))MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAIVISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKP SEQ ID NO: 2(nucleotide sequence for RSV G truncated CX3C aa 1-86 + 155-206 codon-optimized (A strain))ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACACTGGAAAGAACCTGGGACACCCTGAACCATCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAGTCTGTGGCCCAGATCACCCTGAGCATCCTGGCCATCGTGATCAGCACCAGCCTGATCATTGCCGCCATCATCTTTATCGCCAGCGCCAACCACAAAGTGACCCCTACCACAGCCATCATCCAGGACGCCACAAGCCAGATCAAGAACACCCCTCCAAGCAAGCCCAACAACGACTTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAATCCTACCTGCTGGGCCATCTGCAAGAGAATCCCCAACAAGAAGCCCGGCAAAAAGACCACCACAAAGCCCACAAAGAAGCCCTAG SEQ ID NO: 3(amino acid sequence for truncated RSV G protein amino acid 1-206 (A strain))MSKNKDQRTTKTLEKTWDTLNHLLFISSCLYKLNLKSIAQITLSILAMIISTSLIIAAIIFIASANHKVTLTTAIIQDATSQIKNTTPTYLTQNPQLGISFSNLSETTSQTTTILASTTPSVKSTLQSTTVKTKNTTTTKIQPSKPTTKQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPSEQ ID NO: 4(nucleotide sequence for truncated RSV G protein amino acid 1-206(A strain))ATGTCCAAAAACAAGGACCAACGCACCACCAAGACACTAGAAAAGACCTGGGACACTCTCAATCATCTATTATTCATATCATCGTGCTTATACAAGTTAAATCTTAAATCTATAGCACAAATCACATTATCCATTCTGGCAATGATAATCTCAACTTCACTTATAATTGCAGCCATCATATTCATAGCCTCGGCAAACCACAAAGTCACACTAACAACTGCAATCATACAAGATGCAACAAGCCAGATCAAGAACACAACCCCAACATACCTCACCCAGAATCCCCAGCTTGGAATCAGCTTCTCCAATCTGTCTGAAACTACATCACAAACCACCACCATACTAGCTTCAACAACACCAAGTGTCAAGTCAACCCTGCAATCCACAACAGTCAAGACCAAAAACACAACAACAACCAAAATACAACCCAGCAAGCCCACCACAAAACAACGCCAAAACAAACCACCAAACAAACCCAATAATGATTTTCACTTTGAAGTGTTCAACTTTGTACCTTGCAGCATATGCAGCAACAATCCAACCTGCTGGGCTATCTGTAAAAGAATACCAAACAAAAAACCTGGAAAGAAAACCACCACCAAGCCCACAAAAAAACCASEQ ID NO: 5(amino acid sequence for RSV G protein 1-206 codon-optimized (A strain))MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAIVISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKP SEQ ID NO: 6(nucleotide sequence for RSV G protein 1-206 codon-optimized (A strain))ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACACTGGAAAGAACCTGGGACACCCTGAACCATCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAGTCTGTGGCCCAGATCACCCTGAGCATCCTGGCCATCGTGATCAGCACCAGCCTGATCATTGCCGCCATCATCTTTATCGCCAGCGCCAACCACAAAGTGACCCCTACCACAGCCATCATCCAGGACGCCACAAGCCAGATCAAGAACACCACACCTACCTACCTGACACAGAACCCTCAGCTGGGCATCAGCCCTAGCAATCCTAGCGAGATCACCTCTCAGATCACCACAATCCTGGCCAGCACAACCCCTGGCGTGAAGTCTACACTGCAGAGCACCACCGTGAAAACGAAGAATACCACCACCACACAGACCCAGCCTAGCAAGCCTACCACCAAGCAGAGACAGAACAAGCCTCCAAGCAAGCCCAACAACGACTTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAATCCTACCTGCTGGGCCATCTGCAAGAGAATCCCCAACAAGAAGCCCGGCAAAAAGACCACCACAAAGCCCACAAAGAAGCCCTAG SEQ ID NO: 7(amino acid sequence for RSV G protein (A strain), NCBI Reference Sequence:NC_001803.1)MSKNKDQRTTKTLEKTWDTLNHLLFISSCLYKLNLKSIAQITLSILAMIISTSLIIAAIIFIASANHKVTLTTAIIQDATSQIKNTTPTYLTQNPQLGISFSNLSETTSQTTTILASTTPSVKSTLQSTTVKTKNTTTTKIQPSKPTTKQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTIKTTKKDLKPQTTKPKEVPTTKPTEKPTINTTKTNIRTTLLTNNTTGNPEHTSQKGTLHSTSSDGNPSPSQVYTTSEYLSQPPSPSNTTNQ SEQ ID NO: 8(nucleotide sequence for RSV G protein (A strain), NCBI ReferenceSequence: NC_001803.1)ATGTCCAAAAACAAGGACCAACGCACCACCAAGACACTAGAAAAGAAATCTATAGCACAAATCACATTATCCATTCTGGCAATGATAATCTCAACTTCACTTATAATTGCAGCCATCATATTCATAGCCTCGGCAAACCACAAAGTCACACTAACAACTGCAATCATACAAGATGCAACAAGCCAGATCAAGAACACAACCCCAACATACCTCACCCAGAATCCCCAGCTTGGAATCAGCTTCTCCAATCTGTCTGAAACTACATCACAAACCACCACCATACTAGCTTCAACAACACCAAGTGTCAAGTCAACCCTGCAATCCACAACAGTCAAGACCAAAAACACAACAACAACCAAAATACAACCCAGCAAGCCCACCACAAAACAACGCCAAAACAAACCACCAAACAAACCCAATAATGATTTTCACTTTGAAGTGTTCAACTTTGTACCTTGCAGCATATGCAGCAACAATCCAACCTGCTGGGCTATCTGTAAAAGAATACCAAACAAAAAACCTGGAAAGAAAACCACCACCAAGCCCACAAAAAAACCAACCATCAAGACAACCAAAAAAGATCTCAAACCTCAAACCACAAAACCAAAGGAAGTACCTACCACCAAGCCCACAGAAAAGCCAACCATCAACACCACCAAAACAAACATCAGAACTACACTGCTCACCAACAATACCACAGGAAATCCAGAACACACAAGTCAAAAGGGAACCCTCCACTCAACCTCCTCCGATGGCAATCCAAGCCCTTCACAAGTCTATACAACATCCGAGTACCTATCACAACCTCCATCTCCATCCAACACAACAAAC CAGTAGSEQ ID NO: 9(amino acid sequence for RSV G protein full length codon-optimized (A strain))MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAIVISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ SEQ ID NO: 10(nucleotide sequence for RSV G protein full length codon-optimized (A strain))ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACACTGGAAAGAACCTGGGACACCCTGAACCATCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAGTCTGTGGCCCAGATCACCCTGAGCATCCTGGCCATCGTGATCAGCACCAGCCTGATCATTGCCGCCATCATCTTTATCGCCAGCGCCAACCACAAAGTGACCCCTACCACAGCCATCATCCAGGACGCCACAAGCCAGATCAAGAACACCACACCTACCTACCTGACACAGAACCCTCAGCTGGGCATCAGCCCTAGCAATCCTAGCGAGATCACCTCTCAGATCACCACAATCCTGGCCAGCACAACCCCTGGCGTGAAGTCTACACTGCAGAGCACCACCGTGAAAACGAAGAATACCACCACCACACAGACCCAGCCTAGCAAGCCTACCACCAAGCAGAGACAGAACAAGCCTCCAAGCAAGCCCAACAACGACTTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAATCCTACCTGCTGGGCCATCTGCAAGAGAATCCCCAACAAGAAGCCCGGCAAAAAGACCACCACAAAGCCCACAAAGAAGCCCACACTGAAAACCACCAAGAAGGACCCCAAGCCTCAGACCACCAAGTCCAAAGAGGTGCCCACCACCAAACCTACCGAGGAACCCACCATCAACACCACTAAGACCAACATCATCACCACACTGCTGACCTCCAACACCACCGGCAATCCTGAACTGACCAGCCAGATGGAAACCTTCCACAGCACCTCCAGCGAGGGCAACCCATCTCCTAGTCAGGTGTCCACCACAAGCGAGTACCCTAGCCAGCCAAGCAGCCCTCCTAACACACCTAGACAGTAGSEQ ID NO: 11(amino acid sequence for RSV GA strain CX4C aa 1-86 + 155-206 codon-optimized)MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAIVISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTPPSKPNNDFHFEVFNFVPCSICSNNPTCWAIACKRIPNKPGKKTTTKPTKKP SEQ ID NO: 12(nucleotide sequence for RSV GA strain CX4C aa 1-86 + 155-206 codon-optimized)ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACACTGGAAAGAACCTGGGACACCCTGAACCATCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAGTCTGTGGCCCAGATCACCCTGAGCATCCTGGCCATCGTGATCAGCACCAGCCTGATCATTGCCGCCATCATCTTTATCGCCAGCGCCAACCACAAAGTGACCCCTACCACAGCCATCATCCAGGACGCCACAAGCCAGATCAAGAACACCCCTCCAAGCAAGCCCAACAACGACTTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAATCCTACCTGCTGGGCCATCGCCTGCAAGAGAATCCCCAACAAGAAGCCCGGCAAAAAGACCACCACAAAGCCCACAAAGAAGCCCTAG SEQ ID NO: 13(amino acid sequence for RSV G protein full length CX4C (A strain))MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAIVISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAIACKRIPNKKPGKKTTTKPTKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ SEQ ID NO: 14(nucleotide sequence for RSV G protein full length CX4C (A strain))ATGAGCAAGAACAAGGACCAGCGGACCGCCAAGACACTGGAAAGAACCTGGGACACCCTGAACCATCTGCTGTTCATCAGCAGCTGCCTGTACAAGCTGAACCTGAAGTCTGTGGCCCAGATCACCCTGAGCATCCTGGCCATCGTGATCAGCACCAGCCTGATCATTGCCGCCATCATCTTTATCGCCAGCGCCAACCACAAAGTGACCCCTACCACAGCCATCATCCAGGACGCCACAAGCCAGATCAAGAACACCACACCTACCTACCTGACACAGAACCCTCAGCTGGGCATCAGCCCTAGCAATCCTAGCGAGATCACCTCTCAGATCACCACAATCCTGGCCAGCACAACCCCTGGCGTGAAGTCTACACTGCAGAGCACCACCGTGAAAACGAAGAATACCACCACCACACAGACCCAGCCTAGCAAGCCTACCACCAAGCAGAGACAGAACAAGCCTCCAAGCAAGCCCAACAACGACTTCCACTTCGAGGTGTTCAACTTCGTGCCCTGCAGCATCTGCAGCAACAATCCTACCTGCTGGGCCATCGCCTGCAAGAGAATCCCCAACAAGAAGCCCGGCAAAAAGACCACCACAAAGCCCACAAAGAAGCCCACACTGAAAACCACCAAGAAGGACCCCAAGCCTCAGACCACCAAGTCCAAAGAGGTGCCCACCACCAAACCTACCGAGGAACCCACCATCAACACCACTAAGACCAACATCATCACCACACTGCTGACCTCCAACACCACCGGCAATCCTGAACTGACCAGCCAGATGGAAACCTTCCACAGCACCTCCAGCGAGGGCAACCCATCTCCTAGTCAGGTGTCCACCACAAGCGAGTACCCTAGCCAGCCAAGCAGCCCTCCTAACACACCTAGACAGTAGSEQ ID NO: 15(amino acid sequence for RSV G truncated CX3C aa 1-86 + 155-206 (B strain))MSKHKNQRTARTLEKTWDTLNHLIVISSCLYRLNLKSIAQIALSVLAMIISTSLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKP SEQ ID NO: 16(nucleotide sequence for RSV G truncated CX3C aa 1-86 + 155-206 (B strain))ATGTCCAAACACAAGAATCAACGCACTGCCAGGACTCTAGAAAAGACCTGGGATACTCTCAATCATCTAATTGTAATATCCTCTTGTTTATACAGATTAAATTTAAAATCTATAGCACAAATAGCACTATCAGTTCTGGCAATGATAATCTCAACCTCTCTCATAATTGCAGCCATAATATTCATCATCTCTGCCAATCACAAAGTTACACTAACAACGGTCACAGTTCAAACAATAAAAAACCACACTGAAAAAAACCCACCAAAAAAACCAAAAGATGATTACCATTTTGAAGTGTTCAACTTCGTTCCCTGTAGTATATGTGGCAACAATCAACTTTGCAAATCCATCTGTAAAACAATACCAAGCAACAAACCAAAGAAGAAACCAACCATCAAACCCACAAACAAACCATAG SEQ ID NO: 17(amino acid sequence for mutated RSV G truncated CX3C aa 1-86 + 155-206(B strain))MSKHKNQRTARTLEKTWDTLNHLIVISSCLYRLNLKSIAQIALSVLAIVISTSLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKPSEQ ID NO: 18(nucleotide sequence for mutated RSV G truncated CX3C aa 1-86 + 155-206(B strain))ATGTCCAAACACAAGAATCAACGCACTGCCAGGACTCTAGAAAAGACCTGGGATACTCTCAATCATCTAATTGTAATATCCTCTTGTTTATACAGATTAAATTTAAAATCTATAGCACAAATAGCACTATCAGTTCTGGCAATCGTGATCTCAACCTCTCTCATAATTGCAGCCATAATATTCATCATCTCTGCCAATCACAAAGTTACACTAACAACGGTCACAGTTCAAACAATAAAAAACCACACTGAAAAAAACCCACCAAAAAAACCAAAAGATGATTACCATTTTGAAGTGTTCAACTTCGTTCCCTGTAGTATATGTGGCAACAATCAACTTTGCAAATCCATCTGTAAAACAATACCAAGCAACAAACCAAAGAAGAAACCAACCATCAAACCCACAAACAAACCATAG SEQ ID NO: 19(amino acid sequence for RSV G protein B strain, NCBIReference Sequence: NC_001781.1)MSKHKNQRTARTLEKTWDTLNHLIVISSCLYRLNLKSIAQIALSVLAMIISTSLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQPTTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVLDTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA SEQ ID NO: 20(nucleotide sequence for RSV G protein B strain, NCBIReference Sequence: NC_001781.1)ATGTCCAAACACAAGAATCAACGCACTGCCAGGACTCTAGAAAAGACCTGGGATACTCTCAATCATCTAATTGTAATATCCTCTTGTTTATACAGATTAAATTTAAAATCTATAGCACAAATAGCACTATCAGTTCTGGCAATGATAATCTCAACCTCTCTCATAATTGCAGCCATAATATTCATCATCTCTGCCAATCACAAAGTTACACTAACAACGGTCACAGTTCAAACAATAAAAAACCACACTGAAAAAAACATCACCACCTACCTTACTCAAGTCCCACCAGAAAGGGTTAGCTCATCCAAACAACCTACAACCACATCACCAATCCACACAAATTCAGCCACAACATCACCCAACACAAAGTCAGAAACACACCACACAACAGCACAAACCAAAGGCAGAACCACCACCTCAACACAGACCAACAAGCCGAGCACAAAACCACGCCTAAAAAATCCACCAAAAAAACCAAAAGATGATTACCATTTTGAAGTGTTCAACTTCGTTCCCTGTAGTATATGTGGCAACAATCAACTTTGCAAATCCATCTGTAAAACAATACCAAGCAACAAACCAAAGAAGAAACCAACCATCAAACCCACAAACAAACCAACCACCAAAACCACAAACAAAAGAGACCCAAAAACACCAGCCAAAACGACGAAAAAAGAAACTACCACCAACCCAACAAAAAAACCAACCCTCACGACCACAGAAAGAGACACCAGCACCTCACAATCCACTGTGCTCGACACAACCACATTAGAACACACAATCCAACAGCAATCCCTCCACTCAACCACCCCCGAAAACACACCCAACTCCACACAAACACCCACAGCATCCGAGCCCTCTACATCAAATTCCACCCAAAATACCCAATCACATGCTTAG SEQ ID NO: 21(amino acid sequence for mutated RSV G protein full length (B strain))MSKHKNQRTARTLEKTWDTLNHLIVISSCLYRLNLKSIAQIALSVLAIVISTSLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQPTTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVLDTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA SEQ ID NO: 22(nucleotide sequence for mutated RSV G protein full length (B strain))ATGTCCAAACACAAGAATCAACGCACTGCCAGGACTCTAGAAAAGACCTGGGATACTCTCAATCATCTAATTGTAATATCCTCTTGTTTATACAGATTAAATTTAAAATCTATAGCACAAATAGCACTATCAGTTCTGGCAATCGTGATCTCAACCTCTCTCATAATTGCAGCCATAATATTCATCATCTCTGCCAATCACAAAGTTACACTAACAACGGTCACAGTTCAAACAATAAAAAACCACACTGAAAAAAACATCACCACCTACCTTACTCAAGTCCCACCAGAAAGGGTTAGCTCATCCAAACAACCTACAACCACATCACCAATCCACACAAATTCAGCCACAACATCACCCAACACAAAGTCAGAAACACACCACACAACAGCACAAACCAAAGGCAGAACCACCACCTCAACACAGACCAACAAGCCGAGCACAAAACCACGCCTAAAAAATCCACCAAAAAAACCAAAAGATGATTACCATTTTGAAGTGTTCAACTTCGTTCCCTGTAGTATATGTGGCAACAATCAACTTTGCAAATCCATCTGTAAAACAATACCAAGCAACAAACCAAAGAAGAAACCAACCATCAAACCCACAAACAAACCAACCACCAAAACCACAAACAAAAGAGACCCAAAAACACCAGCCAAAACGACGAAAAAAGAAACTACCACCAACCCAACAAAAAAACCAACCCTCACGACCACAGAAAGAGACACCAGCACCTCACAATCCACTGTGCTCGACACAACCACATTAGAACACACAATCCAACAGCAATCCCTCCACTCAACCACCCCCGAAAACACACCCAACTCCACACAAACACCCACAGCATCCGAGCCCTCTACATCAAATTCCACCCAAAATACCCAATCACATGCTTAGSEQ ID NO: 23(amino acid sequence for RSV A strain F protein, DS-Cav1 codon-optimized)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNSEQ ID NO: 24(nucleotide sequence for RSV A strain F protein, DS-Cav1 codon-optimized)cggaaggcccatgaggccagttaattaagaggtaccggatccgccaccATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAAACAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCTAGACGCGAGCTGCCTCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTGCAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCTTCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCCTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGTGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGActcgagctcatggcgcgcctaggccttgacggccttccgSEQ ID NO: 25(coding sequence for RSV A strain F protein, DS-Cav1 codon-optimized)ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAAACAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCTAGACGCGAGCTGCCTCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTGCAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCTTCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCCTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGTGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGASEQ ID NO: 26(amino acid sequence for RSV A strain F protein, SC-DM codon-optimized)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNSEQ ID NO: 27(nucleotide sequence for RSV A strain F protein, SC-DM codon-optimized)cggaaggcccatgaggccagttaattaagaggtaccggatccgccaccATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAATCAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCAGGCTAGA GGC AGC GGA AGCGGA CGG TCCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTCAAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGActcgagctcatggcgcgcctaggccttgacggccttccgSEQ ID NO: 28(coding sequence for RSV A strain F protein, SC-DM codon-optimized)ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAATCAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCAGGCTAGA GGC AGC GGA AGC GGA CGG TCCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTCAAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGA SEQ ID NO: 29(amino acid sequence for RSV A strain F protein, SC-TM codon-optimized)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKIKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGSGSGRSLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDQFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNSEQ ID NO: 30(nucleotide sequence for RSV A strain F protein, SC-TM codon-optimized)cggaaggcccatgaggccagttaattaagaggtaccggatccgccaccATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAATCAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCAGGCTAGA GGC AGC GGAAGC GGA CGG TCCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTCAAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACCAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGActcgagctcatggcgcgcctaggccttgacggccttccg SEQ ID NO: 31(coding sequence for RSV A strain F protein, SC-TM codon-optimized)ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAATCAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCAGGCTAGA GGC AGC GGA AGC GGA CGG TCCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTCAAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACCAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTG ASEQ ID NO: 32(amino acid sequence for RSV F truncated, amino acid 496-574 codon-optimized (A strain))MELLILKANAITTILTAVTFCFASGQNITEEFYQSNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN SEQ ID NO: 33(coding nucleotide sequence for RSV F truncated, amino acid 496-574 codon-optimized (A strain))ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGA SEQ ID NO: 34(amino acid sequence for RSV F protein, NCBI Reference Sequence:NC_001803.1)MELPILKTNAITAILAAVTLCFASSQNITEEFYQTTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKSAVTELQLLMQSTPATNNRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPLAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTINIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN SEQ ID NO: 35(nucleotide sequence for RSV F protein, NCBI Reference Sequence:NC_001803.1)ATGGAGTTGCCAATCCTCAAAACAAATGCAATTACCGCAATCCTTGCTGCAGTCACACTCTGTTTTGCTTCCAGTCAAAACATCACTGAAGAATTTTATCAAACAACATGCAGTGCAGTCAGCAAAGGCTATCTTAGTGCTCTAAGAACTGGTTGGTATACTAGTGTTATAACTATAGAATTAAGTAATATCAAGGAAAATAAGTGTAATGGAACAGACGCTAAGGTAAAATTGATAAAACAAGAATTAGATAAATATAAAAGTGCTGTAACAGAATTGCAGTTGCTCATGCAAAGCACACCGGCAACCAACAATCGAGCCAGAAGAGAACTACCAAGGTTTATGAATTATACACTCAACAATACCAAAAATACCAATGTAACATTAAGCAAGAAAAGGAAAAGAAGATTTCTTGGCTTTTTGTTAGGTGTTGGATCTGCAATCGCCAGTGGCATTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAACAAAATCAAAAGTGCTCTACTATCCACAAACAAGGCTGTAGTCAGCTTATCAAATGGAGTTAGTGTCTTAACCAGCAAAGTGTTAGACCTCAAAAACTATATAGATAAACAGTTGTTACCTATTGTGAACAAGCAAAGCTGTAGCATATCAAACATTGAAACTGTGATAGAGTTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGGGAATTTAGTGTTAATGCAGGTGTAACTACACCTGTAAGCACTTATATGTTAACAAATAGTGAATTATTATCATTAATCAATGATATGCCTATAACAAATGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAGACAGCAAAGTTACTCTATCATGTCCATAATAAAGGAGGAAGTCTTAGCATATGTAGTACAATTACCACTATATGGTGTAATAGATACACCTTGTTGGAAACTGCACACATCCCCTCTATGTACAACCAACACAAAGGAAGGGTCCAACATCTGTTTAACAAGAACCGACAGAGGATGGTACTGTGACAATGCAGGATCAGTATCTTTCTTCCCACTAGCTGAAACATGTAAAGTTCAATCGAATCGAGTATTTTGTGACACAATGAACAGTTTAACATTACCAAGTGAAGTAAATCTCTGCAACATTGACATATTCAACCCCAAATATGATTGCAAAATTATGACTTCAAAAACAGATGTAAGCAGCTCCGTTATCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACAGCATCCAATAAAAATCGTGGAATCATAAAGACATTTTCTAACGGGTGCGATTATGTATCAAATAAGGGGGTTGACACTGTGTCTGTAGGTAATACATTATATTATGTAAATAAGCAAGAAGGCAAAAGTCTCTATGTAAAAGGTGAACCAATAATAAATTTCTATGACCCATTAGTGTTCCCCTCTGATGAATTTGATGCATCAATATCTCAAGTCAATGAGAAGATTAACCAGAGCCTAGCATTTATTCGTAAATCCGATGAATTATTACATAATGTAAATGCTGGTAAATCCACCATAAATATCATGATAACTACTATAATTATAGTGATTATAGTAATATTGTTATCATTAATTGCCGTTGGACTGCTCCTATACTGCAAGGCCAGAAGCACACCAGTCACACTAAGCAAGGATCAACTGAGTGGTATAAATAATATTGCATTTAGTAACTAAAACGGGTGCGATTATGTATCAAATAAGGGGGTTGACACTGTGTCTGTAGGTAATACATTATATTATGTAAATAAGCAAGAAGGCAAAAGTCTCTATGTAAAAGGTGAACCAATAATAAATTTCTATGACCCATTAGTGTTCCCCTCTGATGAATTTGATGCATCAATATCTCAAGTCAATGAGAAGATTAACCAGAGCCTAGCATTTATTCGTAAATCCGATGAATTATTACATAATGTAAATGCTGGTAAATCCACCATAAATATCATGATAACTACTATAATTATAGTGATTATAGTAATATTGTTATCATTAATTGCCGTTGGACTGCTCCTATACTGCAAGGCCAGAAGCACACCAGTCACACTAAGCAAGGATCAACTGAGTGGTATAAATAATATTGCATTTAGTAACTAA SEQ ID NO: 36(amino acid sequence for full length RSV F protein (A strain), NCBI ReferenceSequence: NC_001803.1)MELPILKTNA ITAILAAVTL CFASSQNITE EFYQTTCSAV SKGYLSALRT GWYTSVITIELSNIKENKCN GTDAKVKLIK QELDKYKSAV TELQLLMQST PATNNRARRE LPRFMNYTLNNTKNTNVTLS KKRKRRFLGF LLGVGSAIAS GIAVSKVLHL EGEVNKIKSA LLSTNKAVVSLSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCSISNIETV IEFQQKNNRL LEITREFSVNAGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYVVQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPLAETCKVQSNRVFCDTM NSLTLPSEVN LCNIDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKTKCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDPLVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAGK STINIMITTI IIVIIVILLSLIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSN SEQ ID NO: 37(amino acid sequence for full length RSV F protein, codon-optimized (A strain))MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNSEQ ID NO: 38 (nucleotide sequence for full length RSV F protein, codon-optimized (A strain))ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCTGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGAAAAACAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCTAGACGCGAGCTGCCTCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCCGTGTCAAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGASEQ ID NO: 39 (amino acid sequence for RSV M protein, NCBI ReferenceSequence: NC_001803.1)METYVNKLHEGSTYTAAVQYNVLEKDDDPASLTIWVPMFQSSMPADLLIKELANVNILVKQISTPNGPSLRVMINSRSAVLAQMPSKFTICANVSLDERSKLAYDVTTPCEIKACSLTCLKSKNMLTTVKDLTMKTLNPTHDIIALCEFENIVTSKKVIIPTYLRSISVRNKDLNTLENITTTEFKNAITNAKIIPYSGLLLVITVTDNKGAFKYIKPQSQFIVDLGAYLEKESIYYVTTNWKHTATRFAIKPMED SEQ ID NO: 40(nucleotide sequence for RSV M protein, NCBI ReferenceSequence: NC_001803.1)ATGGAAACATACGTGAACAAACTTCACGAAGGCTCCACATACACAGCTGCTGTTCAATACAATGTCTTAGAAAAAGACGATGACCCTGCATCACTTACAATATGGGTGCCCATGTTCCAATCATCCATGCCAGCAGATTTACTTATAAAAGAACTAGCTAATGTCAACATACTAGTGAAACAAATATCCACACCCAATGGACCTTCATTAAGAGTCATGATAAACTCAAGAAGTGCAGTGCTAGCACAAATGCCCAGCAAATTTACCATATGTGCCAATGTGTCCTTGGATGAAAGAAGCAAGCTGGCATATGATGTAACCACACCCTGTGAAATCAAGGCATGTAGTCTAACATGCCTAAAATCAAAAAATATGTTAACTACAGTTAAAGATCTCACTATGAAAACACTCAACCCAACACATGACATCATTGCTTTATGTGAATTTGAAAATATAGTAACATCAAAAAAAGTCATAATACCAACATACCTAAGATCCATCAGTGTCAGAAATAAAGATCTGAACACACTTGAAAATATAACAACCACTGAATTCAAAAATGCCATCACAAATGCAAAAATCATCCCTTACTCAGGATTACTGTTAGTCATCACAGTGACTGACAACAAAGGAGCATTCAAATACATAAAGCCACAAAGTCAATTTATAGTAGATCTTGGAGCTTACCTAGAAAAAGAAAGTATATATTATGTTACAACAAATTGGAAGCACACAGCTACACGATTTGCAATCAAACCCATGGAAGATTAA SEQ ID NO: 41(amino acid sequence for RSV M protein codon-optimized (A strain))MFQSSMPADLLIKDSTYTAAVQYNVLEKDDDPASLTIWVPMFQSSMPADLLIKELANVNILVKQISTPKGPSLRVMINSRSAVLAQMPSKFTICANVSLDERSKLAYDVTTPCEIKACSLTCLKSKNMLTTVKDLTMKTLNPTHDIIALCEFENIVTSKKVIIPTYLRSISVRNKDLNTLENITTTEFKNAITNAKIIPYSGLLLVITVTDNKGAFKYIKPQSQFIVDLGAYLEKESIYYVTTNWKHTATRFAIKPMED SEQ ID NO: 42(nucleotide sequence for RSV M protein codon-optimized (A strain))ATGTTCCAGAGCAGCATGCCCGCCGACCTGCTGATCAAAGACAGCACCTACACAGCCGCCGTGCAGTACAACGTGCTGGAAAAGGACGACGACCCCGCCAGCCTGACCATCTGGGTGCCCATGTTCCAGAGCAGCATGCCCGCCGACCTGCTGATCAAAGAACTGGCCAACGTGAACATCCTGGTCAAGCAGATCAGCACCCCCAAGGGCCCCAGCCTGAGAGTGATGATCAACAGCCGCAGCGCCGTGCTGGCCCAGATGCCCAGCAAGTTCACCATCTGCGCCAACGTGTCCCTGGACGAGCGGAGCAAGCTGGCCTACGACGTGACCACCCCCTGCGAGATCAAGGCCTGCAGCCTGACCTGCCTGAAGTCCAAGAACATGCTGACCACCGTGAAGGACCTGACCATGAAGACCCTGAACCCCACCCACGACATCATTGCCCTGTGCGAGTTCGAGAACATCGTGACCAGCAAGAAAGTGATCATCCCCACCTACCTGCGGAGCATCAGCGTGCGGAACAAGGACCTGAACACCCTGGAAAACATCACCACCACCGAGTTCAAGAACGCCATTACCAACGCCAAGATCATCCCCTACAGCGGCCTGCTGCTGGTCATCACCGTGACCGACAACAAGGGCGCCTTCAAGTACATCAAGCCCCAGAGCCAGTTCATCGTGGACCTGGGCGCCTACCTGGAAAAAGAATCCATCTACTACGTCACCACCAACTGGAAGCACACCGCCACCAGATTCGCCATCAAGCCCATGGAAGATTGA SEQ ID NO: 43(amino acid sequence for RSV M2-1 protein, NCBI ReferenceSequence: NC_001803.1)MSRRNPCKFEIRGHCLNGKRCHFSHNYFEWPPHALLVRQNFMLNRILKSMDKSIDTLSEISGAAELDRTEEYALGVVGVLESYIGSINNITKQSACVAMSKLLTELNSDDIKKLRDNEEPNSPKIRVYNTVISYIESNRKNNKQTIHLLKRLPADVLKKTIKTTLDIHKSITINNPKESTVSDINDHAKNNDTTSEQ ID NO: 44 (nucleotide sequence for RSV M2-1 protein, NCBI ReferenceSequence: NC_001803.1)ATGTCACGAAGGAATCCTTGCAAATTTGAAATTCGAGGTCATTGCTTGAATGGTAAGAGGTGTCATTTTAGTCATAATTATTTTGAATGGCCACCCCATGCACTGCTTGTAAGACAAAACTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGCATCGATACTTTATCAGAAATAAGTGGAGCTGCAGAGTTGGACAGAACAGAAGAGTATGCCCTCGGTGTAGTTGGAGTGCTAGAGAGTTATATAGGATCTATAAATAATATAACTAAACAATCAGCATGTGTTGCCATGAGCAAACTCCTCACTGAACTCAACAGTGATGACATCAAAAAACTGAGGGACAATGAAGAGCCAAATTCACCCAAGATAAGAGTGTACAATACTGTCATATCATATATTGAAAGCAACAGGAAAAACAATAAACAAACTATCCATCTGTTAAAAAGATTGCCAGCAGACGTATTGAAGAAAACCATAAAAACCACATTGGATATCCACAAGAGCATAACCATCAATAACCCAAAAGAATCAACTGTTAGTGATATAAACGACCATGCCAAAAATAATGATACTACCTGA SEQ ID NO: 45(amino acid sequence for RSV M2-1 protein codon-optimized (A strain))MSRRNPCKFEIRGHCLNGKRCHFSHNYFEWPPHALLVRQNFMLNRILKSMDKSIDTLSEISGAAELDRTEEYALGVVGVLESYIGSINNITKQSACVAMSKLLTELNSDDIKKLRDNEELNSPKIRVYNTVISYIESNRKNNKQTIHLLKRLPADVLKKTIKNTLDIHKSITINNPKESTVSDTNDHAKNNDTT SEQ ID NO: 46(nucleotide sequence for RSV M2-1 protein codon-optimized (A strain))ATGAGCCGGCGGAACCCCTGCAAGTTCGAGATCCGGGGCCACTGCCTGAACGGCAAGCGGTGCCACTTCAGCCACAACTACTTCGAGTGGCCCCCTCACGCCCTGCTGGTGCGCCAGAACTTCATGCTGAACCGGATCCTGAAGTCCATGGACAAGAGCATCGACACCCTGAGCGAGATCAGCGGAGCTGCCGAGCTGGACCGGACCGAGGAATATGCCCTGGGCGTGGTGGGAGTGCTGGAAAGCTACATCGGCAGCATCAACAACATCACCAAGCAGAGCGCCTGCGTGGCCATGAGCAAGCTGCTGACCGAGCTGAACAGCGACGACATCAAGAAGCTGCGGGACAACGAGGAACTGAACAGCCCCAAGATCCGGGTGTACAACACCGTGATCAGCTACATCGAGAGCAACCGGAAGAACAACAAGCAGACCATCCATCTGCTGAAGCGGCTGCCCGCCGACGTGCTGAAGAAAACCATCAAGAACACCCTGGACATCCACAAGTCCATCACCATCAACAACCCCAAAGAAAGCACCGTGTCCGACACCAACGACCACGCCAAGAACAACGACACCACCTGASEQ ID NO: 47 (amino acid sequence for RSV P protein, NCBI ReferenceSequence: NC_001803.1)MEKFAPEFHGEDANNRATKFLESIKGKFTSPKDPKKKDSIISVNSIDIEVTKESPITSNSTIINPTNETDDTVGNKPNYQRKPLVSFKEDPTPSDNPFSKLYKETIETFDNNEEESSYSYEEINDQTNDNITARLDRIDEKLSEILGMLHTLVVASAGPTSARDGIRDAMVGLREDMIEKIRTEALMTNDRLEAMARLRNEESEKMAKDTSDEVSLNPTSEKLNNLLEGNDSDNDLSLDDF SEQ ID NO: 48(nucleotide sequence for RSV P protein, NCBI ReferenceSequence: NC_001803.1)ATGGAAAAGTTTGCTCCTGAATTCCATGGAGAAGATGCAAACAACAGAGCTACCAAATTCCTAGAATCAATAAAGGGCAAATTCACATCACCTAAAGATCCCAAGAAAAAAGATAGTATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAGAAAGCCCTATAACATCAAATTCAACCATTATAAACCCAACAAATGAGACAGATGATACTGTAGGGAACAAGCCCAATTATCAAAGAAAACCTCTAGTAAGTTTCAAAGAAGACCCTACGCCAAGTGATAATCCCTTTTCAAAACTATACAAAGAAACCATAGAAACATTTGATAACAATGAAGAAGAATCTAGCTATTCATATGAAGAAATAAATGATCAGACAAACGATAATATAACAGCAAGATTAGATAGGATTGATGAAAAATTAAGTGAAATACTAGGAATGCTTCACACATTAGTAGTAGCGAGTGCAGGACCTACATCTGCTCGGGATGGTATAAGAGATGCCATGGTTGGTTTAAGAGAAGACATGATAGAAAAAATCAGAACTGAAGCATTAATGACCAATGACAGACTAGAAGCTATGGCAAGACTCAGGAATGAGGAAAGTGAAAAGATGGCAAAAGACACATCAGATGAAGTGTCTCTCAATCCAACATCAGAGAAATTGAACAACCTGTTGGAAGGGAATGATAGTGACAATGATCTATCACTTGATGATTTCTGA SEQ ID NO: 49(amino acid sequence for RSV P protein codon-optimized (A strain))MEKFAPEFHGEDANNRATKFLESIKGKFTSPKDPKKKDSIISVNSIDIEVTKESPITSNSTIINPTNETDDTAGNKPNYQRKPLVSFKEDPTPSDNPFSKLYKETIETFDNNEEESSYSYEEINDQTNDNITARLDRIDEKLSEILGMLHTLVVASAGPTSARDGIRDAMIGLREEMIEKIRTEALMTNDRLEAMARLRNEESEKMAKDTSDEVSLNPTSEKLNNLLEGNDSDNDLSLEDF SEQ ID NO: 50(nucleotide sequence for RSV P protein codon-optimized (A strain))ATGGAAAAGTTCGCCCCCGAGTTCCACGGCGAGGACGCCAACAACCGGGCCACCAAGTTTCTGGAATCCATCAAGGGCAAGTTCACCAGCCCCAAGGACCCCAAGAAGAAGGACAGCATCATCAGCGTGAACAGCATCGACATCGAAGTGACCAAAGAGAGCCCCATCACCAGCAACAGCACCATCATCAACCCCACCAACGAGACAGACGACACCGCCGGCAACAAGCCCAACTACCAGCGGAAGCCCCTGGTGTCCTTCAAAGAGGACCCCACCCCCAGCGACAACCCCTTCAGCAAGCTGTACAAAGAGACAATCGAGACATTCGACAACAACGAGGAAGAGAGCAGCTACAGCTACGAGGAAATCAACGACCAGACCAACGACAACATCACCGCCAGACTGGACCGGATCGACGAGAAGCTGAGCGAGATCCTGGGCATGCTGCACACCCTGGTGGTGGCCTCTGCCGGCCCTACAAGCGCCAGAGATGGCATCCGGGACGCCATGATCGGCCTGCGGGAAGAGATGATCGAGAAGATCCGGACCGAGGCCCTGATGACCAACGACCGGCTGGAAGCCATGGCCCGGCTGCGGAACGAGGAATCCGAGAAGATGGCCAAGGACACCAGCGACGAGGTGTCCCTGAACCCCACCTCTGAGAAGCTGAACAACCTGCTGGAAGGCAACGACAGCGACAACGACCTGAGCCTGGAAGATTTCTGA

1. A virus like particle (VLP) comprising a respiratory syncytial virus(RSV) M protein and an RSV M2-1 protein.
 2. The VLP of claim 1, whereinthe VLP comprises one or more additional RSV proteins.
 3. The VLP ofclaim 1, wherein the VLP comprises an RSV F protein.
 4. The VLP of claim3, wherein the RSV F protein is selected from a group consisting of apre-fusion form of the RSV F protein, a post-fusion form of the RSV Fprotein, and a carbonyl terminal portion of the RSV F protein.
 5. TheVLP of claim 3, wherein the RSV F protein comprises a sequence selectedfrom the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO:29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:
 37. 6.The VLP of claim 3, wherein the carbonyl terminal portion of the RSV Fprotein comprises a sequence of SEQ ID NO:
 32. 7. The VLP of claim 1,wherein the VLP comprises an RSV G protein.
 8. The VLP of claim 7,wherein the RSV G protein is from RSV group A or RSV group B.
 9. The VLPof claim 7, wherein the RSV G protein comprises a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, and SEQ ID NO:
 21. 10. The VLP of claim 1,wherein the VLP comprises a recombinant RSV G protein.
 11. The VLP ofclaim 10, wherein the recombinant RSV G protein comprises atransmembrane domain of an RSV G protein and a central conserved domainof the RSV G protein.
 12. A vaccine comprising the VLP of claim
 1. 13.The vaccine of claim 12, further comprising an adjuvant.
 14. A method ofinducing immunity to RSV infection or at least one symptom thereof in asubject, comprising administering one or more effective doses of thevaccine of claim
 12. 15. The method of claim 14, wherein the one or moreeffective doses of the vaccine are administered to the subject via aroute that is selected from the group consisting of an intramuscularroute, a subcutaneous route, an intradermal route, an oraladministration, a nasal administration, and inhalation.
 16. A virus likeparticle (VLP) comprising a respiratory syncytial virus (RSV) M protein,an RSV P protein, an RSV F protein, and an RSV G protein.
 17. The VLP ofclaim 16, wherein the RSV F protein is selected from a group consistingof a pre-fusion form of the RSV F protein, a post-fusion form of the RSVF protein, and a carbonyl terminal portion of the RSV F protein.
 18. TheVLP of claim 16, wherein the RSV F protein comprises a sequence selectedfrom the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO:29, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:
 37. 19.The VLP of claim 17, wherein the carbonyl terminal portion of the RSV Fprotein comprises a sequence of SEQ ID NO:
 32. 20. The VLP of claim 16,wherein the RSV G protein is from RSV group A or RSV group B.
 21. TheVLP of claim 16, wherein the RSV G protein comprises a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO:
 21. 22. The VLP of claim16, wherein the RSV G protein is a recombinant RSV G protein.
 23. TheVLP of claim 22, wherein the recombinant RSV G protein comprises atransmembrane domain of the RSV G protein and a central conserved domainof the RSV G protein.
 24. A vaccine comprising the VLP of claim
 16. 25.The vaccine of claim 24, further comprising an adjuvant.
 26. A method ofinducing immunity to RSV infection or at least one symptom thereof in asubject, comprising administering one or more effective doses of thevaccine of claim
 24. 27. The method of claim 26, wherein the one or moreeffective doses of the vaccine are administered to the subject via aroute that is selected from the group consisting of an intramuscularroute, a subcutaneous route, an intradermal route, an oraladministration, a nasal administration, and inhalation.